Registration Dossier
Registration Dossier
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EC number: 271-668-0 | CAS number: 68603-62-3
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 July 2017 - 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 28 April 2017 - 21 March 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study was conducted in accordance with international guidelines and in accordance with GLP. The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day dietary study in the Crl:CD(SD) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
- Principles of method if other than guideline:
- The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day oral gavage study in the Crl:WI(Han) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Homogenous suspension formed in corn oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspended in corn oil prior to administration
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as a liquid
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A
OTHER SPECIFICS: N/A - Species:
- rat
- Strain:
- other: CRL:WI(Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 10-12 weeks
- Weight at study initiation: Males: 285.8 - 368.4 g; Females: 182.2 - 236.9 g
- Fasting period before study: Not reported
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of five by sex. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum): Animals had ad libitum access to 5LF2 EU Rodent Diet (International Product Supplies Ltd., London, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels that may have interfered with achieving the objective of the study
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels that may have interfered with achieving the objective of the study.
- Acclimation period: 14-22 days
DETAILS OF FOOD AND WATER QUALITY: Asdescribed above.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12:12
IN-LIFE DATES: From: 23 May 2017 To: 13 June 2017 - Route of administration:
- oral: gavage
- Details on route of administration:
- Test article formulations (excluding the control group) were stirred upon arrival to the animal room from at least 30 minutes prior to, and continuously throughout, dosing.
Formulations were dosed within 3 hours of preparation.
The test article was administered orally by gavage. Animals were dosed once daily for up to 14 consecutive days, excluding the day of necropsy. A dose volume of 10 mL/kg was used. Dose volumes were based on the most recent individual body weight for each animal. - Vehicle:
- corn oil
- Details on oral exposure:
- - PREPARATION OF DOSING SOLUTIONS:
Test article formulations (excluding the control group) were stirred upon arrival to the animal room from at least 30 minutes prior to, and continuously throughout, dosing. Formulations were dosed within 3 hours of preparation.
Method of preparation not reported in range-finder.
- DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A
- VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item formed homogenous formulation in corn oil. Guideline recommended vehicle.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no. (if required): Batch numbers MKBP7039V and MKBZ9899V
- Purity: Not reported, assumed 100 % - Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- N/A
- Duration of treatment / exposure:
- 14 days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 750 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The initial dose level was based on the findings from an acute toxicity study in the rat, where a single dose of 2000 mg/kg was well tolerated (Study 8361291).
- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A - Positive control:
- N/A
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weely
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded once weekly during the predose phase; on Days 1 and 4 (all animals) and 8, 11, and 14 (Control, 500 and 750 mg/kg/day groups only) of the dosing phase; and before each necropsy
FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No - Other examinations:
- N/A
- Statistics:
- Statistical analyses were not performed.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Among surviving animals, stained skin, wet fur, salivation, and thinning fur were observed. Minimal salivation was observed in one male administered 500 mg/kg/day, one male administered 750 mg/kg/day, and one female administered 750 mg/kg/day. Yellow or orange staining of skin (abdomen, legs, nose, and urogenital area), wet fur around the urogenital area and fore legs were observed for animals administered 500 or 750 mg/kg/day, and thinning fur around the forelegs and urogenital area was observed for some animals administered 750 mg/kg/day.
Postdose observations included mouth rubbing, paddling, subdued/sluggish behavior, semi-closed eyes, raised hair, and salivation for animals administered ≥500 mg/kg/day, and reduced activity for animals administered ≥750 mg/kg/day. Mouth rubbing was also observed for controls; however, the duration and frequency of mouth rubbing was higher for test article-treated animals, compared with controls. Mouth rubbing, salivation, paddling, and raised hair were noted immediately upon return to the cage and up to 4 hours postdose. Subdued/sluggish behavior and reduced activity were observed within 30 minutes to 1 hour postdose. At the end of the day, raised hair, salivation, and mouth rubbing continued to be observed for animals administered ≥750 mg/kg/day; sluggish behavior was noted for one female administered 500 mg/kg/day; and reduced activity and sluggish behavior were observed for animals administered 1000 mg/kg/day. On the following morning, the only postdose observations were raised hair and sluggish activity for animals administered 1000 mg/kg/day. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Test article-related mortality was observed for animals administered 1000 mg/kg/day. Two females administered 1000 mg/kg/day were sacrificed in a moribund condition on Day 3 or 6, respectively. Prior to death, one animal was noted as hunched and subdued/sluggish; had loose feces, semi-closed eyes, and brown or yellow stained skin (urogenital area, anus, muzzle, nose, and front paws); and lost 31.7 g of body weight (Days 1 to 3). The second animal was moderately thin; had vocalization, raised hair, yellow stained skin, and wet fur (abdomen, urogenital area); and lost 22.2 g body weight (Days 1 to 6). All remaining animals administered 1000 mg/kg/day, were sacrificed on Day 7 of the dosing phase due to continued body weight losses and clinical observations. Clinical observations for these animals included one or more of the following: hunched, yellow stained skin (urogenital area, inguinal region, abdomen), and wet fur (urogenital area).
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Test article-related body weight losses were observed for the interval of Days 1 to 4 for males administered ≥500 mg/kg/day. For males administered 1000 mg/kg/day, body weight losses continued to be observed for individual animals, until their sacrifice on Day 7. Lower mean body weight gains continued to be observed during the next two intervals (Days 4 to 8 and 8 to 11) for males administered 500 mg/kg/day, and for all remaining intervals of the dosing phase for males administered 750 mg/kg/day. Compared with controls, a higher mean body weight gain was noted for the last dosing phase interval (Days 11 to 14) for males administered 500 mg/kg/day. Compared with controls, overall body weight gain was 60% and ~100% lower, and Day 14 mean body weight was 6% and 16% lower, for males administered 500 or 750 mg/kg/day, respectively.
Test article-related body weight losses were observed for the interval of Days 1 to 4 for females administered ≥500 mg/kg/day. For all subsequent intervals during the dosing phase, mean body weight gain for females administered 500 or 750 mg/kg/day was generally comparable with that of controls therefore indicating the body weight effect was reversed. For females administered 1000 mg/kg/day, body weight losses continued to be observed for individual animals, until their sacrifice on Day 7. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Compared with controls, dose-dependent decreases in mean food consumption were noted during all dosing phase intervals (Days 1 to 4, 4 to 8, 8 to 11, and 11 to 14) for males administered ≥500 mg/kg/day. Decreased food consumption for these animals correlated with body weight loss or decreased body weight gain.
Compared with controls, dose-dependent decreases in mean food consumption were noted for the interval of Days 1 to 4 for females administered ≥500 mg/kg/day. For all subsequent intervals during the dosing phase, mean food consumption for females administered 500 or 750 mg/kg/day was comparable with that of controls. For the interval of Days 4 to 6, females administered 1000 mg/kg/day continued to have low food consumption. Decreased food consumption correlated with body weight loss or decreased body weight gain. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- Compared with controls, mean water consumption was generally higher during the dosing phase for animals administered ≥500 mg/kg/day, except on Days 1 to 2 and
2 to 3, when mean water consumption was minimally lower for animals administered 1000 mg/kg/day. This decrease correlated with general debilitation noted for these animals. - Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- not examined
- Description (incidence and severity):
- N/A
- Clinical biochemistry findings:
- not examined
- Description (incidence and severity):
- N/A
- Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Behaviour (functional findings):
- not examined
- Description (incidence and severity):
- N/A
- Immunological findings:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Group mean unadjusted epididymis (-5 and -12%) and testes (-4 and -10%) weights were lower for males administered 500 or 750 mg/kg/day, compared with concurrent controls. However, group mean body weight ratios for these organs were higher than for controls; therefore, these decreased unadjusted organ weights were considered to be related to body weight losses and not directly test article related.
- Description (incidence and severity):
- Macroscopic observations were recorded in the stomach, liver, kidney, mesenteric lymph nodes, lungs, adrenal, skin, uterus, and testes. Liver findings were observed at all dose levels, and kidney and stomach findings were observed in animals administered ≥750 mg/kg/day.
Findings in the stomach included distension, dark or thick mucosa, irregular surface, pale appearance, or raised focus/area for two females administered 1000 mg/kg/day and three females administered 750 mg/kg/day.
Findings in the kidney included pale, mottled appearance, or pelvic dilatation in three males administered 1000 mg/kg/day and two males administered 750 mg/kg/day.
Liver findings included large, mottled, or pale appearance in two males administered 1000 mg/kg/day (Animals R0301 and R0304), two males administered 750 mg/kg/day, one female administered 750 mg/kg/day, and one female administered 1000 mg/kg/day. Incidence of findings in the kidney, liver, and stomach exhibited dose-response.
Based on the incidence, findings in the stomach, liver, and kidney were considered potentially test article related. Findings in other tissues were observed in a single animal or lacked a dose-response; therefore, a relationship to the test article could not be ruled out. - Neuropathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings: non-neoplastic:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings: neoplastic:
- not examined
- Description (incidence and severity):
- N/A
- Other effects:
- not examined
- Description (incidence and severity):
- N/A
- Conclusions:
- In a 14-day OECD 422 dose range-finding study, daily oral gavage administration of 500, 750 or 1000 mg/kg/day Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) to male and female rats was not tolerated following 750 or 1000 mg/kg/day administration. Based on the findings of this 14 day repeated dose study, dose levels of 500, 300 and 100 mg/kg/day were recommended for the following OECD 422 study.
- Executive summary:
OECD 422 Range Finder (2018) - The purpose of this test was to assess the systemic toxicity potential of Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) following repeated exposure to 20 male and 20 female Crl:WI(Han) strain rats by dietary administration at dose levels of 500, 750 and 1000 ppm for up to 14 days. The findings would be used to select suitable dose levels for a subsequent combined repeat dose toxicity study with reproductive/ developmental toxicity screening study (OECD 422).
A summary of adult responses to the test item are described below;
Mortality- Two females administered 1000 mg/kg/day were sacrificed in a moribund condition on Day 3 or 6, respectively, and all remaining animals administered 1000 mg/kg/day were sacrificed on Day 7 of the dosing phase due to adverse clinical observations and body weight loss.
Clinical signs- Generalised clinical observations which included stained skin, wet fur, salivation, thinning fur, subdued/sluggish behavior, salivation, paddling, raised hair and mouth rubbing were observed in the 500 and 750 mg/kg/day test groups. Additionally, in the 750 mg/kg/day group reduced activity was observed.
A few postdose observations continued to be observed at the end of the day for animals administered 750 mg/kg/day, whereas postdose observations were generally resolved by the end of each day of dosing for animals administered 500 mg/kg/day.
Bodyweight and food consumption- Administration of 500 or 750 mg/kg/day resulted in initial body weight losses, with correlating decreased food consumption. Decreased body weight gain and food consumption continued to be observed until the end of the dosing phase for males administered 750 mg/kg/day; whereas decreased body weight gain was reversed by the end of the dosing phase for animals administered 500 mg/kg/day and decreased food consumption was reversed by the end of the dosing phase for females administered 500 mg/kg/day.
Necropsy- Macroscopic findings were observed in the stomach and kidney for both sexes administered ≥750 mg/kg/day and in the liver for both sexes administered ≥500 mg/kg/day.
Organ weights- Compared with concurrent controls, group mean unadjusted epididymis (-5 and -12%) and testes (-4 and -10%) weights were lower for males administered 500 or 750 mg/kg/day, which were related to body weight losses and not directly test article related.
Based on these findings, a dose level of 750 mg/kg/day may not be tolerated for a longer duration of dosing due to findings of consistently decreased body weight gain and food consumption, in conjunction with the clinical observations of subdued behavior and reduced activity.
Dose levels of 500, 300 and 100 mg/kg/day are recommended for the following OECD 422 study.
Table 3 Mean body weights and body weight gains/ losses
Sex / Day |
Bodyweight (g) |
|||
Control |
500 ppm |
750 ppm |
1000 ppm |
|
MALES |
||||
Pre-dose Day 1 |
298.5 |
303.5 |
279.6 |
303.5 |
Pre-dose Day 8 |
- |
- |
307.6 |
323.7 |
Day 1 |
333.4 |
335.4 |
312.6 |
328.2 |
Day 4 (change from Day 1-4) |
339.7 (6.3) |
332.4 (-3.0) |
306.6 (-6.0) |
313.6 (-14.6) |
Day 8 (change from Day 4-8) |
354.5 (14.8) |
341.2 (8.8) |
307.9 (1.2) |
- (-) |
Day 11 (change from Day 8-11) |
363.4 (8.9) |
341.2 (0.1) |
314.4 (6.5) |
- (-) |
Day 14 (change from Day 11-14) |
370.9 (7.5) |
350.4 (9.2) |
312.5 (-1.9) |
- (-) |
Change from Day 1-14 |
37.5 |
15.0 |
-0.1 |
- |
FEMALES |
||||
Pre-dose Day 1 |
199.2 |
206.6 |
188.3 |
205.3 |
Pre-dose Day 8 |
- |
- |
201.2 |
215.3 |
Day 1 |
205.3 |
211.6 |
200.2 |
218.0 |
Day 4 (change from Day 1-4) |
205.9 (0.6) |
203.8 (-7.8) |
193.7 (-6.5) |
199.6 (-14.0) |
Day 8 (change from Day 4-8) |
212.4 (6.5) |
212.4 (8.6) |
202.9 (9.2) |
- (-) |
Day 11 (change from Day 8-11) |
214.4 (2.0) |
213.0 (0.6) |
205.1 (2.3) |
- (2.3) |
Day 14 (change from Day 11-14) |
209.9 (-4.5) |
214.1 (1.1) |
210.2 (5.0) |
- (-) |
Change from Day 1-14 |
4.6 |
2.4 |
10.0 |
- |
Table 4 Unadjusted organ weights (relative to body weight %)
Doses (ppm) |
Control |
7500 |
11000 |
3000 |
Control |
7500 |
11000 |
3000 |
Males |
Females |
|||||||
Number of animals/group |
5 |
5 |
5 |
0 |
0 |
0 |
0 |
0 |
BODY WEIGHT (termination weight for toxicity males) |
||||||||
Weight (g) |
358.4 |
333.9 |
297.4 |
- |
- |
- |
- |
- |
EPIDIDMYSIS (termination weight for toxicity males) |
||||||||
Weight (g) |
1.279 |
1.214 |
1.128 |
- |
- |
- |
- |
- |
% body weight |
0.3583 |
0.3639 |
0.3789 |
- |
|
|
|
|
TESTIS (termination weight for toxicity males) |
||||||||
Weight (g) |
3.594 |
3.445 |
3.252 |
- |
- |
- |
- |
- |
% body weight |
1.0044 |
1.0370 |
1.0989 |
- |
- |
- |
- |
- |
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 July 2017 - 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- other: CRL:WI(Han)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:
Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported, assume yes
- Age at study initiation:
10-11 weeks old
- Weight at study initiation:
Males: 288.6 - 391.8 g; females: 173.2 - 235.3 g
- Fasting period before study:
No
- Housing:
During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation, and with their litter during the lactation phase. Males were returned to group-housing after the pairing phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum):
Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum):
Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
- Acclimation period:
Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for at least 14 days prior to initiation of dosing (males) or 7 days prior to initiation of smearing (females).
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 ºC
- Humidity (%):
30-70 % RH
- Air changes (per hr):
15-20
- Photoperiod (hrs dark / hrs light):
12:12
IN-LIFE DATES: From: 26 July 2017 To: 25 September 2017 - Route of administration:
- oral: gavage
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
##
- VEHICLE
- Justification for use and choice of vehicle (if other than water):
Homogenous stable solutions were prepared in corn oil, a guideline recommended vehicle.
- Concentration in vehicle:
##
- Amount of vehicle (if gavage):
5 mL/kg
- Lot/batch no. (if required):
MKBP7039V, MKBZ9899V and MKCC0462
- Purity: Assumed pure corn oil - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Single 5.0 mL aliquot formulation samples were collected from the main in-life experiment study for each animal group at the respective concentrations (0, 20.0, 60.0 and 100.0 mg/mL) on the 26th July 2017 (week 1 of the in-life phase), 9th August 2017 (week 3 of the in-life phase) and 30th August 2017 (week 6 of the in-life phase) for analysis.
A validated gradient elution reversed-phase high-performance liquid chromatographic method with ultraviolet detection (HPLC-UV) was employed for determination of formulation stability and homogeneity, and concentration analyses of formulations administered during the study.
No test item was observed in the control group samples. Measured concentrations from the test item groups were found to be in the range of 89.2 - 102.3 % of nominal values. - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: Up to 15 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 (GD 0) of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility for up to an additional 5 days.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Females were housed individually during gestation, and with their litter during the lactation phase.
- Any other deviations from standard protocol: No - Duration of treatment / exposure:
- Males: 42 consecutive days (2 weeks prior to pairing; during pairing; and until the day before necropsy)
Females: 61 days (2 weeks prior to pairing [pre-pairing phase]; during the pairing phase; and until Lactation Day (LD) 13, inclusive, or 25 days post-coitum for females which did not litter and were sent to necropsy on LD 14 or 26 days post coitum). Some females were not dosed on LD 0 if they were observed to be starting or just completed parturition. - Frequency of treatment:
- Daily
- Duration of test:
- Males: 42 consecutive days (2 weeks prior to pairing; during pairing; and until the day before necropsy)
Females: 61 days (2 weeks prior to pairing [pre-pairing phase]; during the pairing phase; and until Lactation Day (LD) 13, inclusive, or 25 days post-coitum for females which did not litter and were sent to necropsy on LD 14 or 26 days post coitum). Some females were not dosed on LD 0 if they were observed to be starting or just completed parturition. - Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10 males and 10 females per treatment group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses selected based on the results of a preliminary range-finder test.
- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
BODY WEIGHT: Yes
- Time schedule: Male body weights were recorded once during acclimation, before dosing on the first day of dosing, at weekly intervals, and before necropsy. Female body weights were recorded once during acclimation; before dosing on the first day of dosing; at weekly intervals prior to pairing and until confirmation of mating; on Gestation Day (GD) 0, 7, 14, and 20; and on LD 1, 4, 13 and 14 (prior to necropsy).
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and LD 0 to 13. Consumption was calculated as g/animal/day.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The amount of water consumed was determined daily during the pre-pairing phase (both sexes). Consumption was calculated as g/animal/day.
OTHER: N/A - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: N/A - Fetal examinations:
- N/A
- Statistics:
- Data for each sex were analyzed separately, unless stated otherwise. Only data collected on or after the first day of dosing were analyzed statistically. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P ≤ 0.05. By default, significant results were reported as *P ≤ 0.05, +P ≤ 0.01, and/or #P ≤ 0.001.
- Indices:
- The duration of gestation
Number of implantation sites
Number of pups born
Number of pups alive on Post Natal Day (PND) 1, 4 (before culling)
% male pups on PND 1
Percent post-implantation loss
Live birth and survival indices PND 1-4
Pup weights
Ano-genital distance (male only)
Thyroid hormones - Historical control data:
- Reffered to but details not reported
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- An observation of vocalizing on handling was noted in one female from each test article treated group from Pre-Pairing Day 4 or 5. One female administered 500 mg/kg/day was noted to have wet fur of the anogenital area and/or abdomen during the pre-pairing phase or GD 7. These observations were attributed to test article.
Incidences of mouth rubbing were noted immediately upon the return to the home cage in up to 4 of 10 females administered 500 mg/kg/day. Salivation was observed from Pre-Pairing Day 8 for some females administered 100, 300, or 500 mg/kg/day and persisted through pairing for occasional females, but no dose response was noted. Salivation was not recorded during gestation or lactation. These observations were attributed to the consistency and palatability of the test article and were considered non-adverse. - Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality:
- no mortality observed
- Description (incidence):
- No maternal female mortality was observed.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weight gain was for females administered 500 mg/kg/day, when compared with controls during late gestation.
Mean body weight during the pre-pairing, gestation, or lactation phases for females administered 100, 300 mg/kg/day were compared with controls and considered unaffected by the test article. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Lower food consumption was noted for males and females administrated 500 mg/kg/day during the first two weeks of dosing. Food consumption was also noted to be lower for 500 mg/kg/day females during late gestation, when compared with controls.
No effect on food consumption was noted following 100 or 300 mg/kg/day administration. - Food efficiency:
- not examined
- Description (incidence and severity):
- N/A
- Description (incidence and severity):
- A test article related effect on water consumption was noted for animals administered 300 or 500 mg/kg/day.
Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, compared with controls, such that at the end of the pre-pairing phase, female water consumption was 38 or 80% greater than controls, respectively. - Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Haematological parameters were unaffected by the test item.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Cholesterol and glucose levels were statisticaly significantly elevated in females administered 300 mg/kg/day. Data from the 500 mg/kg/day female was also elevated in comparison to the control however the data was excluded from statistical analysis as only 1 female was analysed.
All other values, including those which attained statistical significance, were considered incidental and not related to Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) as no dose relationship was noted and/or values were within background ranges. - Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- Female changes during the assessments included piloerection and salivation, which were generally dose related and behavior changes predominantly characterized by paw flicking (fore paws). A statistically significant decreased number of rears were noted on Lactation Day (LD) 1 and 7 for females 300 mg/kg/day, compared with controls. A lower number was also recorded on LD 1 for females administered 500 mg/kg/day; these females were early decedents due to litter loss and, therefore, no data are available for LD 7.
- Immunological findings:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Organ weight parameters were unaffected by the test item.
- Gross pathological findings:
- not specified
- Description (incidence and severity):
- Small thymus was recorded in one female administered 300 mg/kg/day.
- Neuropathological findings:
- no effects observed
- Description (incidence and severity):
- Locomotor activity was unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In the liver, centrilobular hepatocyte hypertrophy was recorded in all examined females administered 300 or 500 mg/kg/day. This finding was characterized by the enlargement of the hepatocytes, a ground glass appearance of the cytoplasm, and slight compression of the adjacent sinusoids; was principally localized within the centrilobular areas, but occasionally also extended more diffusely to midzonal and periportal regions in affected animals; and correlated generally with increased group mean adjusted liver weights and the macroscopic finding of dark/mottled discoloration.
In the thyroid, follicular cell hypertrophy was recorded in four females administered 300 mg/kg/day and one female administered 500 mg/kg/day and was characterized by follicular cells with increased amounts of cytoplasm, follicular epithelium that ranged from flat to cuboidal or columnar and follicles with decreased amounts of colloid. - Histopathological findings: neoplastic:
- no effects observed
- Description (incidence and severity):
- No tumours were observed.
- Other effects:
- not examined
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- No incident of abortion was observed.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- Implantation loss (%) was unaffected by the test item.
- Total litter losses by resorption:
- not specified
- Description (incidence and severity):
- One female treated at 500 mg/kg/day did not litter and was found to have 10 implantation sites with no evidence of foetus in the uterus. This is indicative of 100 % early resorption.
The report did not discuss if this was a test item related response. - Early or late resorptions:
- not specified
- Description (incidence and severity):
- One female treated at 500 mg/kg/day did not litter and was found to have 10 implantation sites with no evidence of foetus in the uterus. This is indicative of 100 % early resorption.
The report did not discuss if this was a test item related response. - Dead fetuses:
- effects observed, treatment-related
- Description (incidence and severity):
- Test article-related effects on litter survival were noted. Eight of 10 females administered 500 mg/kg/day were sent to necropsy between LD 0 and 2 due to total litter loss. In all females that successfully littered, up to 100% of pups were still born, with three litters which contained still born pups only. The litter death was attributed to the test article and was adverse.
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Pregnancy duration was not effected by the test item.
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- One control female and one female administered 300 mg/kg/day were sent to necropsy on Gestation Day (GD) 26 having failed to produce a litter. At necropsy, the animals were found to be non-pregnant. The non-pregnant finding for these animals was considered incidental, and no relationship to dose was noted.
- Details on maternal toxic effects:
- No test article-related effects on clinical or postdose observations were noted, although lower body weight gain and food consumption was noted during late gestation following 500 mg/kg/day administration.
Some markers of toxicity were observed. The FOB observations included piloerection, salivation, and paw flicking (fore paws), which increased in a dose dependent manner. A decreased number of rears was also noted during the lactation phase for females 300 mg/kg/day, compared with controls. Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, at 38 or 80% greater than the controls, respectively. Cholesterol and glucose levels were also elevated in females administered 300 mg/kg/day. However, the macroscopic and/or microscopic observations noted in the kidney of males were not apparent in females.
Microscopic observations included epidermal hyperplasia and/or hyperkeratosis in the nonglandular stomach, which were considered consistent with a chronic irritation-type reaction.
Test article-related effects on litter survival were noted. Eight of 10 females administered 500 mg/kg/day were sent to necropsy between LD 0 and 2 due to total litter loss. In all females that successfully littered, up to 100% of pups were still born, with three litters which contained still born pups only. The litter death was attributed to the test article and was adverse.
Hepatocyte hypertrophy in the liver and the associated increase in TSH and thyroid follicular cell hypertrophy were less pronounced in females than males; nevertheless, it is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites and is considered rat specific. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- behaviour (functional findings)
- body weight and weight gain
- clinical biochemistry
- dead fetuses
- food consumption and compound intake
- water consumption and compound intake
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A test article-related effect on body weight gain was noted for pups from litters of dams administered 300 or 500 mg/kg/day, compared with controls.
Only two litters survived to PND 1 and only one litter survived to PND 13 due to the number of total litter losses between PND 0 and 2 observed for litters of dams administered 500 mg/kg/day. Of the few surviving litters, a reduction in body weight was noted, compared with controls. Where statistical evaluation was possible (PND 1), this reduction attained a statistical significance. - Reduction in number of live offspring:
- effects observed, treatment-related
- Description (incidence and severity):
- Nine of 10 females administered 500 mg/kg/day produced a litter. Five litters were recorded to have live pups at birth, with a single litter surviving to scheduled necropsy; in this litter only 4 of 12 pups alive on PND 1. For females administered 500 mg/kg/day, between 22 to 100% of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. One litter contained still born pups only, and, excluding one litter which survived to PND 13, all others had total litter death by PND 2.
An effect was observed for the live birth index of females administered 300 mg/kg/day compared with controls (83.4% compared to 100% for control), but postnatal survival was comparable with control values. - Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- effects observed, treatment-related
- Description (incidence and severity):
- A reduction in mean body weight values from litters of dams administered 300 mg/kg/day was noted from PND 1 to 13 (between 80 to 86%). This attained statistical significance in males (PND1, 4, 7 and 13) and females (PND 1, 4 and 7).
No test article-related effect was noted in litters of dams administered 100 mg/kg/day. - Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- One litter from a control dam and one litter from a dam administered 300 mg/kg/day died by PND 2. The 300 mg/kg/day animal had 12 pups in total, two still born pups and all remaining pups found dead with no milk evident in the stomach on PND 0. The control animal had 1 pup in total, which was noted to be cold to touch, weak, unfed, and was found dead on PND 2. The deaths were considered incidental as they were noted in controls or occurred as a single incident in the group, with no other effects on pup survival.
- External malformations:
- no effects observed
- Description (incidence and severity):
- Only one 500 mg/kg/day litter survived to PND 4 due the high incidence of litter death; as such, this may have been incidental and not related to test article even though values were lower. No effect on ano-genital distance was observed in litters of dams administered 100 or 300 mg/kg/day.
No nipple/areolae were observed for males surviving to PND 13. - Skeletal malformations:
- not examined
- Visceral malformations:
- not examined
- Other effects:
- not examined
- Details on embryotoxic / teratogenic effects:
- Excluding the total litter death noted for the litters of dams administered 500 mg/kg/day, no other test article-related pup clinical observations were noted.
A test article-related effect on body weight and body weight gain was noted for pups from litters of dams administered 300 or 500 mg/kg/day, compared with controls.
Only two litters survived to PND 1 and only one litter survived to PND 13 due to the number of total litter losses between PND 0 and 2 observed for litters of dams administered 500 mg/kg/day. Of the few surviving litters, a reduction in body weight was noted, compared with controls. Where statistical evaluation was possible (PND 1), this reduction attained a statistical significance.
A reduction in mean body weight values from litters of dams administered 300 mg/kg/day was noted from PND 1 to 13 (between 80 to 86%). This attained statistical significance in males, females, and/or combined values on all body weight occasions. A reduction in combined body weight gain of 2.9 g was also noted between PND 1 to 13.
No external malformations or effects on thyroid hormone levels was noted at any treatment level.
In summary, no test article-related effect was noted in litters of dams administered 100 mg/kg/day. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- reduction in number of live offspring
- fetal/pup body weight changes
- changes in litter size and weights
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 300 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Conclusions:
- No test article-related changes were noted in the litters following parental administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for developmental toxicity was established as 100 mg/kg/day.
- Executive summary:
OECD 422 (2018) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Amines, C12-12-branched alkyl, docdecylbenzenesulfonates (1:1) was administered to 30 male and 30 female Crl:WI(Han) strain rats by oral gavage administration at dose levels of 100, 300 and 500 mg/kg/day for up to 6 weeks. In addition, 10 male and 10 female were used as control rats and were treated with the corn oil vehicle only.
A summary of developmental toxicity the test item are described below;
Litters and offspring
Nine of 10 females administered 500 mg/kg/day produced a litter, of which one entire litter was stillborn. For females administered 500 mg/kg/day, between 22 to 100% of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. Eight litters were recorded with live pups at birth, with total litter losses noted by PND 2 for four of these litters, with only one litter surviving to scheduled necropsy on PND 13. The litter death was attributed to the test article and was considered adverse.
A test article-related effect on the live birth index was observed following 300 mg/kg/day administration (83.4% compared to 100% for controls), however, post-implantation survival index and pup survival indices were comparable with controls and sex ratio was unaffected
A reduction in mean body weight values from litters of dams administered 300 mg/kg/day was noted from PND 1 to 13 (between 80 to 86%). This attained statistical significance in male pups (PND 1, 4, 7 and 13) and female pups (PND 1, 4 and 7). No test article-related effect was noted in litters of dams administered 100 mg/kg/day.
Ano-genital distance, pup thyroid hormone levels, and male nipple/areolae count were unaffected by the test article.
Endpoint discussion
No test article-related changes were noted in the litters following parental administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for developmental toxicity was established as 100 mg/kg/day.
Table 5 Summary of Parturition and Litter Data
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
|
Duration of gestation (days) |
Mean |
23.0 |
23.1 |
22.6 |
23.1 |
N |
9 |
10 |
9 |
9 |
|
No. of implantation sites |
Mean |
11.3 |
10.6 |
12.3 |
12.3 |
N |
9 |
10 |
9 |
9 |
|
No. of pups born |
Mean |
10.7 |
9.2 |
11.7 |
10.0 |
N |
9 |
10 |
9 |
9 |
|
No. of pups alive at Post-Natal Day 1 |
Mean |
0.7 |
9.2 |
9.7 |
0.8*** |
N |
9 |
10 |
9 |
9 |
|
% male pups at PND 1 |
Mean |
49.4 |
47.4 |
59.0 |
29.2 |
N |
9 |
10 |
8 |
2 |
|
No. of pups alive at PND 4 before culling |
Mean |
10.6 |
9.1 |
10.9 |
2.0 |
N |
9 |
10 |
8 |
1 |
|
No. of pups culled at PND 4 |
Mean |
1.9 |
1.0 |
1.6 |
0.0 |
N |
8 |
10 |
8 |
1 |
|
No. of pups alive after PND 4 culling |
Mean |
10.0 |
8.1 |
9.3 |
4.0 |
N |
8 |
10 |
8 |
1 |
|
No. of pups alive at PND 7 |
Mean |
10.0 |
8.0 |
9.3 |
4.0 |
N |
8 |
10 |
8 |
1 |
|
No. of pups alive at PND 13 |
Mean |
10.0 |
8.0 |
9.3 |
4.0 |
N |
8 |
10 |
8 |
1 |
|
*** p<0.001
Table 6 Summary of pup survival
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
|
Post-implantation survival index % |
Mean |
94.7 |
87.7 |
94.6 |
81.4 |
N |
9 |
10 |
9 |
9 |
|
Live birth index % |
Mean |
100.0 |
100.0 |
83.4 |
7.4*** |
N |
9 |
10 |
9 |
9 |
|
Survival index PND 1-4 % |
Mean |
88.9 |
95.0 |
100.0 |
50.0 |
N |
9 |
10 |
8 |
2 |
|
Survival index PND 4-7 % |
Mean |
100.0 |
99.0 |
100.0 |
100.0 |
N |
8 |
10 |
8 |
1 |
|
Survival index PND 7-13 % |
Mean |
100.0 |
100.0 |
100.0 |
100.0 |
N |
8 |
10 |
8 |
1 |
|
*** p <0.001
Table 7 Summary of pup clinical observations
Clinical observation |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Bruised tip of tail |
- |
- |
1 |
- |
Cold |
1 |
- |
1 |
1 |
Found dead |
1 |
1 |
2 |
8 |
Hemorrhagic area to face |
- |
1 |
- |
- |
Missing, presumed cannibalized |
- |
- |
1 |
5 |
Thinning fur |
- |
1 |
- |
- |
Total litter loss |
1 |
- |
1 |
8 |
Umbilical cord attached |
- |
- |
1 |
- |
Unfed |
1 |
1 |
- |
- |
Weak |
1 |
1 |
- |
- |
Table 8 Summary of pup body weights
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
||
Post-Natal Day 1 |
Male |
Adjusted mean |
7.2 |
7.1 |
5.9** |
5.1*** |
Mean |
7.2 |
7.1 |
5.9 |
5.1 |
||
N |
8 |
10 |
8 |
2 |
||
Female |
Adjusted mean |
6.6 |
6.9 |
5.6** |
5.3* |
|
Mean |
6.6 |
6.8 |
5.7 |
5.3 |
||
N |
9 |
10 |
8 |
2 |
||
Combined |
Adjusted mean |
6.8 |
7.0 |
5.7** |
5.3* |
|
Mean |
6.8 |
7.0 |
5.8 |
5.3 |
||
N |
9 |
10 |
8 |
2 |
||
Post-Natal Day 4 |
Male |
Adjusted mean |
11.0 |
11.0 |
8.8*** |
- |
Mean |
10.9 |
11.2 |
8.7 |
7.6 |
||
N |
8 |
9 |
8 |
1 |
||
Female |
Adjusted mean |
10.3 |
10.5 |
8.4* |
- |
|
Mean |
10.3 |
10.6 |
8.4 |
9.0 |
||
N |
8 |
10 |
8 |
1 |
||
Combined |
Adjusted mean |
10.6 |
10.7 |
8.6** |
- |
|
Mean |
10.6 |
10.7 |
8.6 |
8.7 |
||
N |
8 |
10 |
8 |
1 |
||
Post-Natal Day 7 |
Male |
Adjusted mean |
16.4 |
16.9 |
13.2** |
- |
Mean |
16.4 |
17.0 |
13.2 |
5.4 |
||
N |
8 |
9 |
8 |
1 |
||
Female |
Adjusted mean |
15.5 |
16.2 |
12.9* |
- |
|
Mean |
15.6 |
16.0 |
13.0 |
7.1 |
||
N |
8 |
10 |
8 |
1 |
||
Combined |
Adjusted mean |
15.9 |
16.4 |
13.0** |
- |
|
Mean |
16.0 |
16.2 |
13.1 |
6.7 |
||
N |
8 |
10 |
8 |
1 |
||
Post-Natal Day 13 |
Male |
Adjusted mean |
28.4 |
29.7 |
24.0** |
- |
Mean |
28.4 |
29.7 |
24.0 |
27.2 |
||
N |
8 |
9 |
8 |
1 |
||
Female |
Adjusted mean |
27.1 |
28.5 |
23.4 |
- |
|
Mean |
27.5 |
28.0 |
23.7 |
29.1 |
||
N |
8 |
10 |
8 |
1 |
||
Combined |
Adjusted mean |
27.5 |
28.8 |
23.5 |
- |
|
Mean |
27.9 |
28.3 |
23.8 |
29.1 |
||
N |
8 |
10 |
8 |
1 |
||
* p<0.05
** p<0.01
***p <0.001
Table 9 Summary of ano-genital distance (mm) at PND 4
|
Male |
Female |
|
Control |
Adjusted mean |
3.6 |
- |
Mean |
3.8 |
2.0 |
|
N |
8 |
8 |
|
100 mg/kg/day |
Adjusted mean |
3.9 |
- |
Mean |
4.1 |
2.0 |
|
N |
9 |
10 |
|
300 mg/kg/day |
Adjusted mean |
4.0 |
- |
Mean |
3.5 |
1.8 |
|
N |
7 |
7 |
|
500 mg/kg/day |
Adjusted mean |
- |
- |
Mean |
3.0 |
1.0 |
|
N |
1 |
1 |
|
Table 10 Summary of male nipples/ areolae at PND 13
|
No. of litter examined |
Count |
Control |
8 |
0 |
100 mg/kg/day |
9 |
0 |
300 mg/kg/day |
8 |
0 |
Table 11 Summary of pup thyroid analysis at PND 13
|
Male |
Female |
Combined |
|||||||
IMT4 (nmol/L) |
TSHI (µIU/mL) |
TT3S (nmol/L) |
IMT4 (nmol/L) |
TSHI (µIU/mL) |
TT3S (nmol/L) |
IMT4 (nmol/L) |
TSHI (µIU/mL) |
TT3S (nmol/L) |
||
Control |
Adjusted mean |
83 |
0.31 |
0.89 |
88 |
0.27 |
0.99 |
86 |
0.29 |
0.94 |
Mean |
11.6 |
0.122 |
0.174 |
12.8 |
0.086 |
0.110 |
11.6 |
0.088 |
0.123 |
|
N |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
|
100 mg/kg/day |
Adjusted mean |
96 |
0.23 |
1.13* |
95 |
0.26 |
1.08 |
95 |
0.24 |
1.12* |
Mean |
10.7 |
0.106 |
0.171 |
15.2 |
0.165 |
0.175 |
11.7 |
0.128 |
0.167 |
|
N |
9 |
9 |
9 |
10 |
10 |
10 |
10 |
10 |
10 |
|
300 mg/kg/day |
Adjusted mean |
90 |
0.19 |
1.00 |
91 |
0.22 |
0.92 |
91 |
0.21 |
0.96 |
Mean |
13.5 |
0.043 |
0.143 |
20.0 |
0.051 |
0.121 |
14.8 |
0.033 |
0.119 |
|
N |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
|
500 mg/kg/day |
Adjusted mean |
87E |
0.17E |
1.18E |
96E |
0.28E |
1.36E |
92E |
0.23E |
1.27E |
Mean |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
N |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
|
IMT4: Imulite Total T4
TSHI: Rapid Thyroid Stimulating Hormone
TT3S: Total T3
E: excluded from dataset
* p<0.05
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 July 2017 to 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- other: CRL:WI(Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:
Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported, assume yes
- Age at study initiation:
10-11 weeks old
- Weight at study initiation:
Males: 288.6 - 391.8 g; females: 173.2 - 235.3 g
- Fasting period before study:
No
- Housing:
During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation, and with their litter during the lactation phase. Males were returned to group-housing after the pairing phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum):
Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum):
Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
- Acclimation period:
Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for at least 14 days prior to initiation of dosing (males) or 7 days prior to initiation of smearing (females).
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 ºC
- Humidity (%):
30-70 % RH
- Air changes (per hr):
15-20
- Photoperiod (hrs dark / hrs light):
12:12
IN-LIFE DATES: From: 26 July 2017 To: 25 September 2017 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- - PREPARATION OF DOSING SOLUTIONS:
##
- VEHICLE
- Justification for use and choice of vehicle (if other than water):
Homogenous stable solutions were prepared in corn oil, a guideline recommended vehicle.
- Concentration in vehicle:
##
- Amount of vehicle (if gavage):
5 mL/kg
- Lot/batch no. (if required):
MKBP7039V, MKBZ9899V and MKCC0462
- Purity: Assumed pure corn oil - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: Up to 15 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 (GD 0) of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility for up to an additional 5 days.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Females were housed individually during gestation, and with their litter during the lactation phase.
- Any other deviations from standard protocol: No - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Single 5.0 mL aliquot formulation samples were collected from the main in-life experiment study for each animal group at the respective concentrations (0, 20.0, 60.0 and 100.0 mg/mL) on the 26th July 2017 (week 1 of the in-life phase), 9th August 2017 (week 3 of the in-life phase) and 30th August 2017 (week 6 of the in-life phase) for analysis.
A validated gradient elution reversed-phase high-performance liquid chromatographic method with ultraviolet detection (HPLC-UV) was employed for determination of formulation stability and homogeneity, and concentration analyses of formulations administered during the study.
No test item was observed in the control group samples. Measured concentrations from the test item groups were found to be in the range of 89.2 - 102.3 % of nominal values. - Duration of treatment / exposure:
- Males: 42 consecutive days (2 weeks prior to pairing; during pairing; and until the day before necropsy)
Females: 61 days (2 weeks prior to pairing [pre-pairing phase]; during the pairing phase; and until LD 13, inclusive, or 25 days post-coitum for females which did not litter and were sent to necropsy on LD 14 or 26 days post coitum). Some females were not dosed on LD 0 if they were observed to be starting or just completed parturition. - Frequency of treatment:
- Daily
- Details on study schedule:
- N/A
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10 males and 10 females per treatment group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses selected based on the results of a preliminary range-finder test.
- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A - Positive control:
- N/A
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
BODY WEIGHT: Yes
- Time schedule: Male body weights were recorded once during acclimation, before dosing on the first day of dosing, at weekly intervals, and before necropsy. Female body weights were recorded once during acclimation; before dosing on the first day of dosing; at weekly intervals prior to pairing and until confirmation of mating; on Gestation Day (GD) 0, 7, 14, and 20; and on LD 1, 4, 13 and 14 (prior to necropsy).
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and LD 0 to 13. Consumption was calculated as g/animal/day.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The amount of water consumed was determined daily during the pre-pairing phase (both sexes). Consumption was calculated as g/animal/day.
OTHER: N/A - Oestrous cyclicity (parental animals):
- Daily vaginal washings were conducted for all females during acclimation (predose) from 1 week after arrival until the day prior to dosing. The stage of estrous was recorded, and only females with regular 4 to 5 day cycles were included on study.
Daily vaginal washings were conducted on females from the start of dosing until the confirmation of mating and on LD 14, prior to necropsy. - Sperm parameters (parental animals):
- Parameters examined in F0 male parental generations:
testis weight, epididymis weight - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed, sexed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups
GROSS EXAMINATION OF DEAD PUPS:
After sacrifice, surplus pups culled on PND 4 had the sex determined; no macroscopic examination was required.
After sacrifice, decedent pups and one pup/sex/litter sacrificed on PND 13 were examined internally. The remaining pups sacrificed were examined externally for gross abnormalities, with particular attention to the external reproductive genitals.
Thyroid glands of pups culled on PND 13 (one pup/sex/litter across groups) were weighed approximately 24 hours post-fixation, and then and preserved in 10% neutral buffered formalin
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: N/A
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: N/A - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals on post-pairing Day 14 after the preliminary evaluation of female data and an overnight period without food.
- Maternal animals: All surviving animals on lactation Day (LD) 14 (those that achieved pregnancy) or Day 26 post coitum (those that did not litter) after an overnight period without food.
GROSS NECROPSY
- Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.
HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 3 were prepared for microscopic examination and weighed, respectively. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring were sacrificed at 13 days of age (study terminus) or at 4 days of age (litter standardisation).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Pups sacrificed were examined externally for gross abnormalities, with particular attention to the external reproductive genitals.
To provide one pooled sample for each litter, where possible, pups culled on PND 4 had blood samples (1 x 0.6 mL) withdrawn for thyroid hormone analysis via decapitation.
To provide two pooled samples for each litter from Groups 1, 2, and 3 (one sample from two males and one sample from two females, where possible), pups sacrificed on PND 13 had blood samples (2 x 0.8 mL) withdrawn for thyroid hormone analysis by cardiac puncture. All pups sacrificed on PND 13 had blood samples (1 x 0.8 mL) withdrawn for thyroid hormone analysis by cardiac puncture.
GROSS NECROPSY
- After sacrifice, decedent pups and one pup/sex/litter sacrificed on PND 13 were examined internally. The remaining pups sacrificed were examined externally for gross abnormalities, with particular attention to the external reproductive genitals.
HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands of pups culled on PND 13 (one pup/sex/litter across groups) were weighed approximately 24 hours post-fixation, and then and preserved in 10% neutral buffered formalin. - Statistics:
- Data for each sex were analyzed separately, unless stated otherwise. Only data collected on or after the first day of dosing were analyzed statistically. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P ≤ 0.05. By default, significant results were reported as *P ≤ 0.05, +P ≤ 0.01, and/or #P ≤ 0.001.
- Reproductive indices:
- Male and female mating
Fecundity
Fertility indices - Offspring viability indices:
- Duration of gestation
Number of implantation sites
Number of pups born
Number of pups alive on PND 1, 4 (before culling)
% male pups on PND 1
Percent post-implantation loss
Live birth and survival indices PND 1-4
Pup weights
Ano-genital distance (males only)
Thyroid hormone - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Urogenital staining and wet abdominal fur were occasionally recorded during Pre Pairing and Pairing for one or two males administered 300 or 500 mg/kg/day. An observation of vocalizing on handling was noted in one female from each test article treated group from Pre-Pairing Day 4 or 5. One female administered 500 mg/kg/day was noted to have wet fur of the anogenital area and/or abdomen during the pre-pairing phase or GD 7. These observations were attributed to test article.
Incidences of mouth rubbing were noted immediately upon the return to the home cage in up to 4 of 10 females administered 500 mg/kg/day. Salivation was observed from Pre-Pairing Day 8 for some females administered 100, 300, or 500 mg/kg/day and persisted through pairing for occasional females, but no dose response was noted. Salivation was not recorded during gestation or lactation. Salivation was also noted for males administered 100, 300, or 500 mg/kg/day, without a dose response, from Pre-Pairing Day 9, 7, and 10, respectively, and until the end of the pre-pairing, through the pairing, and into the post pairing phases. These observations were attributed to the consistency and palatability of the test article and were considered non-adverse.
All other observations, including fur staining, fur loss, sores/lesions, and tail damage, were considered incidental and not related to the test article as they were also observed in control animals, showed no dose relationship, and/or are commonly observed in animals of this age and strain. - Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality:
- not specified
- Description (incidence):
- One male administered 500 mg/kg/day was found dead on Study Day 16 (Pairing Day 1). No adverse clinical observation were noted prior to the animal being found dead, and no specific cause of demise could be determined from the tissues examined. The death, therefore, had an uncertain relationship to the test article.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Test article-related lower mean body weight gain was recorded for males administered 300 or 500 mg/kg/day. Mean male body weight gain was statistically significantly reduced during the pre pairing phase for males administered 500 mg/kg/day and was 67% lower than controls by the end of the pre-pairing phase. Thereafter, mean body weight gain for males was similar to or slightly lower than controls, but this did not attain statistical significance. Overall, the mean male body weight gain at completion of the study was statistically significantly reduced at 57% of the control value for males administered 500 mg/kg/day. While individual incidences did not attain statistical significance for males administered 300 mg/kg/day, the overall gain for the study showed a statistically significantly reduction (77% of the control value). Mean body weight was unaffected in males administered 100 mg/kg/day, compared with controls.
Body weight gain was also lower for females administered 500 mg/kg/day, when compared with controls during late gestation.
Mean body weight during the pre-pairing, gestation, or lactation phases for females administered 100, 300 mg/kg/day were compared with controls and considered unaffected by the test article. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Lower food consumption was noted for males and females administrated 500 mg/kg/day during the first two weeks of dosing. Food consumption was also noted to be lower for 500 mg/kg/day females during late gestation, when compared with controls.
No effect on food consumption was noted following 100 or 300 mg/kg/day administration. - Food efficiency:
- not examined
- Description (incidence and severity):
- N/A
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- A test article related effect on water consumption was noted for animals administered 300 or 500 mg/kg/day.
Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, compared with controls, such that at the end of the pre-pairing phase, male water consumption was 67 or 100% greater than controls, respectively, and female water consumption was 38 or 80% greater than controls, respectively. - Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Hematology parameters were unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- A statistically significant elevation in glucose levels was noted in males administered 500 mg/kg/day, and a statistically significant increase in cholesterol, calcium, and inorganic phosphate levels was noted in males administered 300 or 500 mg/kg/day, compared with control. Cholesterol and glucose levels were also elevated in females administered 300 mg/kg/day.
A statistically significant increase in TSH was noted in males administered 300 mg/kg/day (155 % of control) and animals administered 500 mg/kg/day (188 and 176 % of control) following approximately 6 weeks of dosing. These values fell outside of background control ranges. - Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean urine volume was increased for males administered 300 or 500 mg/kg/day, compared with controls (121 and 86 % of control values, respectively); the increase attained statistical significance for males administered 300 mg/kg/day.
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- During the assessments, moderate salivation, piloerection, and changes in body/head posture were observed for males in all test article treated groups, compared with controls. In addition, a dose related increase in ataxia was noted, and high stepping, walking on toes, and slow deliberate movements were noted for animals administered 300 or 500 mg/kg/day, although a decrease in activity was not observed in these groups, compared with controls. A statistically significant increased number of rears were noted on Pre-Pairing Day 6 for males administered 300 or 500 mg/kg/day and Post-Pairing Day 12 for males administered 500 mg/kg/day, compared with controls.
Female changes during the assessments included piloerection and salivation, which were generally dose related and behavior changes predominantly characterized by paw flicking (fore paws). A statistically significant decreased number of rears were noted on LD 1 and 7 for females 300 mg/kg/day, compared with controls. A lower number was also recorded on LD 1 for females administered 500 mg/kg/day; these females were early decedents due to litter loss and, therefore, no data are available for LD 7. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Locomotor activity was unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- A range of Amines-related findings were recorded in the liver, thyroid, kidney, and nonglandular stomach of animals administered 100, 300, or 500 mg/kg/day.
In the liver, centrilobular hepatocyte hypertrophy was recorded in two males administered 100 mg/kg/day, all males and all examined females administered 300 or 500 mg/kg/day. This finding was characterized by the enlargement of the hepatocytes, a ground glass appearance of the cytoplasm, and slight compression of the adjacent sinusoids; was principally localized within the centrilobular areas, but occasionally also extended more diffusely to midzonal and periportal regions in affected animals; and correlated generally with increased group mean adjusted liver weights and the macroscopic finding of dark/mottled discoloration.
In the thyroid, follicular cell hypertrophy was recorded in one male administered 100 mg/kg/day, most males and four females administered 300 mg/kg/day and all males and one female administered 500 mg/kg/day and was characterized by follicular cells with increased amounts of cytoplasm, follicular epithelium that ranged from flat to cuboidal or columnar and follicles with decreased amounts of colloid. This finding correlated generally with increased group mean adjusted thyroid weights and with the macroscopic finding of dark discoloration in one male administered 500 mg/kg/day.
In the kidney, nephropathy was recorded in all examined males administered Amines, which was characterized by multifocal areas of tubular basophilia, inflammatory foci dominated by lymphocytes and/or plasma cells, and granular casts principally located at the corticomedullary junction, but occasionally also in cortical and/or papillary tubules. In addition, hyaline droplets in the cytoplasm of tubular epithelial cells of males administered 100, 300 or 500 mg/kg/day were recorded with higher gradings, compared with concurrent controls. This finding was characterized by focally extensive areas of brightly eosinophilic aggregates of cytoplasmic droplets principally located in the superficial cortex, but multifocally extending towards to the corticomedullary junction. Both of these microscopic findings correlated generally with increased group mean adjusted kidney weights and with the macroscopic finding of mottled and/or pale discoloration.
In addition, in the nonglandular stomach, epithelial hyperplasia and orthokeratotic hyperkeratosis were recorded in one male administered 300 mg/kg/day and four males administered 500 mg/kg/day; these correlated generally with mucosal thickening or the presence of a raised focus, macroscopically - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- No adverse effects on the estrous cycles were observed following test article administration.
An increased length of estrous cycles was recorded for females administered 500 mg/kg/day in pre-pairing. However, as the pre-pairing mean cycle length was reduced in females administered 300 mg/kg/day, the mean number of estrous cycle lengths was unaffected values were within historical background control ranges this observation is considered to be incidental. - Reproductive function: sperm measures:
- not examined
- Description (incidence and severity):
- N/A
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Mating and fertility were unaffected by test article administration.
The majority of animals mated within the first four days of pairing. Two females administered 300 mg/kg/day and two females administered 500 mg/kg/day did not mate within one estrus (4/5 days). However, mating did occur and the females achieved pregnancy, therefore, the delay in mating was considered to be of little toxicological significance.
One control female and one female administered 300 mg/kg/day were sent to necropsy on GD 26 having failed to produce a litter. At necropsy, the animals were found to be non-pregnant. The non-pregnant finding for these animals was considered incidental, and no relationship to dose was noted. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- reproductive performance
- Key result
- Critical effects observed:
- no
- Clinical signs:
- not examined
- Description (incidence and severity):
- N/A
- Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality:
- not examined
- Description (incidence):
- N/A
- Body weight and weight changes:
- not examined
- Description (incidence and severity):
- N/A
- Food consumption and compound intake (if feeding study):
- not examined
- Description (incidence and severity):
- N/A
- Food efficiency:
- not examined
- Description (incidence and severity):
- N/A
- Water consumption and compound intake (if drinking water study):
- not examined
- Description (incidence and severity):
- N/A
- Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- not examined
- Description (incidence and severity):
- N/A
- Clinical biochemistry findings:
- not examined
- Description (incidence and severity):
- N/A
- Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Behaviour (functional findings):
- not examined
- Description (incidence and severity):
- N/A
- Immunological findings:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- not examined
- Description (incidence and severity):
- N/A
- Gross pathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Neuropathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings: non-neoplastic:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings: neoplastic:
- not examined
- Description (incidence and severity):
- N/A
- Other effects:
- not examined
- Description (incidence and severity):
- N/A
- Details on results:
- N/A
- Reproductive function: oestrous cycle:
- not examined
- Description (incidence and severity):
- N/A
- Reproductive function: sperm measures:
- not examined
- Description (incidence and severity):
- N/A
- Reproductive performance:
- not examined
- Description (incidence and severity):
- N/A
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Excluding the total litter death noted for the litters of dams administered 500 mg/kg/day, no other test article-related pup clinical observations were noted.
- Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- A test article-related effect on live birth index for dams administered 500 or 300 mg/kg/day and pup survival for litters of dams administered 500 mg/kg/day was adversely affected.
Nine of 10 females administered 500 mg/kg/day produced a litter. Five litters were recorded to have live pups at birth, with a single litter surviving to scheduled necropsy; in this litter only 4 of 12 pups alive on PND 1. For females administered 500 mg/kg/day, between 22 to 100% of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. One litter contained still born pups only, and, excluding one litter which survived to PND 13, all others had total litter death by PND 2.
An effect was observed for the live birth index of females administered 300 mg/kg/day compared with controls (83.4% compared to 100% for control), but postnatal survival was comparable with control values.
One litter from a control dam and one litter from a dam administered 300 mg/kg/day died by PND 2. Another animal had 12 pups in total, two still born pups and all remaining pups found dead with no milk evident in the stomach on PND 0. A third animal had 1 pup in total, which was noted to be cold to touch, weak, unfed, and was found dead on PND 2. The deaths were considered incidental as they were noted in controls or occurred as a single incident in the group, with no other effects on pup survival. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A test article-related effect on body weight and body weight gain was noted for pups from litters of dams administered 300 or 500 mg/kg/day, compared with controls.
Only two litters survived to PND 1 and only one litter survived to PND 13 due to the number of total litter losses between PND 0 and 2 observed for litters of dams administered 500 mg/kg/day. Of the few surviving litters, a reduction in body weight was noted, compared with controls. Where statistical evaluation was possible (PND 1), this reduction attained a statistical significance.
A reduction in mean body weight values from litters of dams administered 300 mg/kg/day was noted from PND 1 to 13 (between 80 to 86%). This attained statistical significance in males, females, and/or combined values on all body weight occasions. A reduction in combined body weight gain of 2.9 g was also noted between PND 1 to 13.
No test article-related effect was noted in litters of dams administered 100 mg/kg/day. - Food consumption and compound intake (if feeding study):
- not examined
- Description (incidence and severity):
- N/A
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- N/A
- Water consumption and compound intake (if drinking water study):
- not examined
- Description (incidence and severity):
- N/A
- Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- not examined
- Description (incidence and severity):
- N/A
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No effect of test article on pup thyroid hormone levels was noted. All values, including the values that attained statistical significance, were considered incidental and not related to the test article as no dose relationship was apparent and/or the values were within background ranges.
- Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Sexual maturation:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- not examined
- Description (incidence and severity):
- N/A
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Compared with concurrent controls, no treatment-related changes to pup thyroid weights on PND 13 were suggestive of effects due to test article administration to the adults.
- Histopathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Other effects:
- not examined
- Behaviour (functional findings):
- not examined
- Description (incidence and severity):
- N/A
- Developmental immunotoxicity:
- not examined
- Description (incidence and severity):
- N/A
- Key result
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- mortality
- body weight and weight gain
- Key result
- Critical effects observed:
- no
- Clinical signs:
- not examined
- Description (incidence and severity):
- N/A
- Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality / viability:
- not examined
- Description (incidence and severity):
- N/A
- Body weight and weight changes:
- not examined
- Description (incidence and severity):
- N/A
- Food consumption and compound intake (if feeding study):
- not examined
- Description (incidence and severity):
- N/A
- Food efficiency:
- not examined
- Description (incidence and severity):
- N/A
- Water consumption and compound intake (if drinking water study):
- not examined
- Description (incidence and severity):
- N/A
- Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- not examined
- Description (incidence and severity):
- N/A
- Clinical biochemistry findings:
- not examined
- Description (incidence and severity):
- N/A
- Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Sexual maturation:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- not examined
- Description (incidence and severity):
- N/A
- Gross pathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Other effects:
- not examined
- Description (incidence and severity):
- N/A
- Behaviour (functional findings):
- not examined
- Description (incidence and severity):
- N/A
- Developmental immunotoxicity:
- not examined
- Description (incidence and severity):
- N/A
- Key result
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 300 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to other toxic effects:
- reproductive effects as a secondary non-specific consequence of other toxic effects
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Conclusions:
- Males - Reproductive performance was not affected by the test article.
Females - Reproductive performance was not affected by the test article
Parturition and litter - Test article-related effects on litter survival were noted. Eight of 10 females administered 500 mg/kg/day were sent to necropsy between LD 0 and 2 due to total litter loss. In all females that successfully littered, up to 100% of pups were still born, with three litters which contained still born pups only. The litter death was attributed to the test article and was adverse. - Executive summary:
OECD 422 (2018) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Amines, C12-12-branched alkyl, docdecylbenzenesulfonates (1:1) was administered to 30 male and 30 female Crl:WI(Han) strain rats by oral gavage administration at dose levels of 100, 300 and 500 mg/kg/day for up to 6 weeks. In addition, 10 male and 10 female were used as control rats and were treated with the corn oil vehicle only.
A summary of adult reproductive responses to the test item are described below;
Males
Reproductive performance was not affected by the test article i.e. no test-article related effects on mating or fertility were noted.
Females and litter
Nine of 10 females administered 500 mg/kg/day produced a litter, of which one entire litter was stillborn. For females administered 500 mg/kg/day, between 22 to 100 % of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. Eight litters were recorded with live pups at birth, with total litter losses noted by PND 2 for four of these litters, with only one litter surviving to scheduled necropsy on PND 13.
The litter death was attributed to the test article and was considered adverse.
A test article-related effect on the live birth index was observed following 300 mg/kg/day administration (83.4% compared to 100% for controls), however, post-implantation survival index and pup survival indices were comparable with controls and sex ratio was unaffected
Endpoint discussion
Test article-related findings observed in males following 500 mg/kg/day administration were generally confined to systemic toxicity in the rat. These findings were considered not to represent an adverse effect in reproduction, and hence, the no observed effect level (NOEL) for reproductive toxicity in males was established as 500 mg/kg/day. The NOEL for females reproductive toxicity was established as 100 mg/kg/day based on the number of litter losses observed in the 500 mg/kg/day test group and
the test article-related effect on the live birth index following 300 mg/kg/day administration.
Mean number of implantation sites was unaffected by test article administration, however, post-implantation survival index was lower in the 500 mg/kg/day dose group, compared with controls, resulting from a total in utero litter loss for one female from this group.
Live birth and pup survival (PND 1-4) indices were adversely affected following 500 mg/kg/day administration. Nine of 10 females from this dose group produced a litter, of which one entire litter was stillborn. For females administered 500 mg/kg/day, between 22 to 100% of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. Eight litters were recorded with live pups at birth, with total litter losses noted by PND 2 for four of these litters, with only one litter surviving to scheduled necropsy on PND 13. From the surviving litter, only 4 of 12 pups were alive on PND 1. Due to the survival of only one litter, assessment of sex ratio (% males) could not be accurately assessed for this dose level.
A test article-related effect on the live birth index was observed following 300 mg/kg/day administration (83.4% compared to 100% for controls), however, post-implantation survival index and pup survival indices were comparable with controls and sex ratio was unaffected.
One 300 mg/kg/day litter and one control litter died by PND 2. Another 300 mg/kg/day female had 12 pups in total, two still born pups and all remaining pups found dead with no milk evident in the stomach on PND 0. Another 300 mg/kg/day female had 1 pup in total, which was noted to be cold to touch, weak, unfed, and was found dead on PND 2. The deaths were considered incidental as they were noted in controls or occurred as a single incident in the group, with no other effects on pup survival.
No test article-related effects were noted in litters following maternal administration of 100 mg/kg/day.
Table 4 Group mean achieved dose rates for each treatment group (in mg/kg bw/day)
Test Group (mg/kg/day) |
Nominal formulation concentration (mg/mL) |
Actual formulation concentration achieved (% of nominal) |
||
Week 1 |
Week 3 |
Week 6 |
||
Control |
0 |
nd |
nd |
nd |
100 |
20 |
95.8 |
97.3 |
102.3 |
300 |
60 |
96.9 |
95.7 |
96.3 |
500 |
100 |
94.8 |
95.9 |
89.2 |
nd: not detected
Table 5 Mean body weights and body weight gains/ losses (n=10 unless stipulated otherwise) (F0)
Sex / Day |
Bodyweight (g) |
|||
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
|
MALES |
||||
Predose |
||||
Day 1 |
301.5 |
317.7 |
313.0 |
305.7 |
Pre-pairing |
||||
Day 1 |
325.1 |
342.6 |
339.0 |
328.4 |
Day 8 |
345.1 |
365.8 |
355.2 |
331.6 |
Day 15 |
363.1 |
382.3 |
370.8 |
341.0 |
Change Day 1-8 |
20.1 |
23.2 |
16.2 |
3.3** |
Change Day 8-15 |
18.0 |
16.5 |
15.5 |
9.3* |
Pairing |
||||
Day 7 |
369.2 |
389.2 |
372.7 |
348.6 (n=9) |
Day 14 |
385.1 |
406.3 |
385.5 |
365.1 (n=9) |
Change Day 7-14 |
15.9 |
17.1 |
12.8 |
16.5 |
Post-pairing |
||||
Day 7 |
400.6 |
420.8 |
396.2 |
374.9 (n=9) |
Day 13 |
407.5 |
424.2 |
402.2 |
378.2 (n=9) |
Change Day 7-13 |
6.8 |
3.4 |
6.0 |
3.3 |
Overall weight change (pre-pairing Day 1 – post-pairing Day 13) |
82.5 |
81.6 |
63.2* |
47.0** |
FEMALES |
||||
Predose |
||||
Day 8 |
190.1 |
189.6 |
208.6 |
217.3 |
Pre-pairing |
||||
Day 1 |
198.1 |
197.9 |
208.6 |
215.2 |
Day 8 |
208.6 |
199.4 |
212.1 |
225.4 |
Day 15 |
217.3 |
200.6 |
209.4 |
219.4 |
Change Day 1-8 |
10.5 |
10.8 |
12.8 |
8.8 |
Change Day 8-15 |
8.6 |
6.5 |
13.3 |
10.0 |
Gestation |
||||
Day 0 |
220.6 (n=9) |
242.0 |
268.4 (n=9) |
326.9 (n=9) |
Day 7 |
217.5 (n=9) |
238.8 |
263.4 (n=9) |
314.7 (n=9) |
Day 14 |
228.0 (n=9) |
247.3 |
277.1 (n=9) |
332.3 (n=9) |
Day 20 |
221.0 (n=9) |
244.6 |
269.1 (n=9) |
311.6 (n=9) |
Change Day 0-7 |
21.4 |
21.3 |
19.4 |
23.7 |
Change Day 7-14 |
26.4 |
24.5 |
29.8 |
24.4 |
Change Day 14-20 |
58.5 |
51.3 |
55.2 |
42.6 |
Lactation |
||||
Day 1 |
252.8 (n=9) |
252.3 |
255.3 (n=8) |
- |
Day 4 |
256.6 (n=8) |
259.4 |
261.6 (n=8) |
- |
Day 13 |
275.5 (n=8) |
274.9 |
286.8 (n=8) |
- |
Day 14 |
258.2 (n=8) |
255.0 |
254.9 (n=8) |
- |
Change Day 1-4 |
3.7 |
7.1 |
6.4 |
- |
Change Day 4-13 |
18.9 |
15.6 |
25.2 |
- |
Change Day 13-14 |
-17.3 |
-20.0 |
-31.9* |
- |
Overall weight change (pre-pairing Day 1 – gestation Day 20) |
128.6 |
116.8 |
134.0 |
110.7 |
* p<0.05, **p<0.01
Table 6 Haematology, clinical chemistry, urinalysis and pathology findings (F0)
Doses (ppm) |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Male |
Female |
|||||||
Pathology (remarkable changes) |
||||||||
Adrenals - pale, moderate - large, slight - pale, slight |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=5) 1 0 0 |
(n=5) 0 1 1 |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=1) 0 0 0 |
Thyroid - dark, slight - pale, slight - small, slight |
(n=10) 0 0 0 |
(n=10) 0 0 0 |
(n=10) 0 0 0 |
(n=9) 1 0 0 |
(n=8) 0 1 1 |
(n=10) 0 0 0 |
(n=8) 0 2 1 |
(n=1) 0 0 0 |
Thymus - dark, minimal - red focus - small, slight |
(n=5) 0 0 0 |
(n=5) 1 0 0 |
(n=5) 0 1 0 |
(n=5) 0 1 0 |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=5) 0 0 1 |
(n=1) 0 0 0 |
Lymph node, mesenteric - red, slight |
(n=5) 0 |
(n=5) 2 |
(n=5) 0 |
(n=5) 1 |
(n=5) 1 |
(n=5) 1 |
(n=5) 1 |
(n=1) 0 |
Liver - mottled, moderate - mottled, slight - pale, moderate - pale, slight |
(n=7) 1 1 0 2 |
(n=5) 0 4 0 1 |
(n=7) 0 3 1 2 |
(n=7) 1 3 0 2 |
(n=5) 0 0 0 0 |
(n=5) 0 0 0 0 |
(n=5) 0 0 0 0 |
(n=1) 0 0 0 0 |
Stomach - raised focus, pale - red, slight - striation, red - thick, minimal - thick, slight |
(n=5) 0 1 0 0 0 |
(n=5) 0 1 0 0 0 |
(n=5) 0 1 2 1 1 |
(n=5) 1 0 2 0 1 |
(n=5) 0 0 0 0 0 |
(n=5) 0 1 0 0 0 |
(n=5) 0 0 0 0 0 |
(n=1) 0 0 0 0 0 |
Colon - distension, slight |
(n=5) 0 |
(n=5) 0 |
(n=5) 1 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=1) 0 |
Lymph node, mandibular - red, moderate |
(n=5) 0 |
(n=5) 1 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=1) 0 |
Kidney - mottled, minimal - mottled, moderate - mottled, slight - pale, moderate - pale, slight - pelvic dilation, minimal - pelvic dilation, slight - red area |
(n=5) 0 0 0 0 0 0 0 0 |
(n=5) 0 0 2 0 2 0 0 1 |
(n=8) 0 1 5 1 0 1 1 0 |
(n=7) 1 1 2 1 0 0 0 0 |
(n=5) 0 0 0 0 0 0 0 0 |
(n=5) 0 0 0 0 0 0 0 0 |
(n=5) 0 0 0 0 0 0 1 0 |
(n=5) 0 0 0 0 0 0 0 0 |
Testis - small, severe |
(n=10) 0 |
(n=10) 1 |
(n=10) 0 |
(n=9) 0 |
n/a |
n/a |
n/a |
n/a |
Epididymis - small, marked |
(n=10) 0 |
(n=10) 1 |
(n=10) 0 |
(n=9) 0 |
n/a |
n/a |
n/a |
n/a |
Skin - fur loss, moderate - fur loss, slight - sore, slight |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=2) 1 0 1 |
(n=2) 0 2 1 |
(n=0) 0 0 0 |
Lung - pale focus |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 1 |
(n=1) 0 |
Tail - kinked |
(n=0) 0 |
(n=0) 0 |
(n=0) 0 |
(n=0) 0 |
(n=2) 2 |
(n=0) 0 |
(n=0) 0 |
(n=0) 0 |
Peyer’s Patch - prominent, moderate |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 1 |
(n=5) 0 |
(n=5) 0 |
(n=1) 0 |
Mammary gland - thick, moderate |
(n=10) 0 |
(n=10) 0 |
(n=10) 0 |
(n=9) 0 |
(n=8) 0 |
(n=10) 1 |
(n=8) 0 |
(n=1) 0 |
* P<0.05, ** P<0.01
# excluded from dataset
Table 7 Absolute and adjusted organ weights (F0)
Doses (mg/kg/day) |
Control |
100 |
300 |
500 |
Control |
100 |
300 |
500 |
MALE |
FEMALE |
|||||||
BODY WEIGHT |
||||||||
Weight (g) |
391.8 |
407.9 |
381.0 |
356.0* |
249.8 |
253.8 |
252.2 |
|
TESTES |
||||||||
- Absolute weight (g) |
3.561 |
3.534 |
3.724 |
3.781 |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.912 |
0.878 |
0.982 |
1.068 |
n/a |
n/a |
n/a |
n/a |
EPIDIDYMIS |
||||||||
- Absolute weight (g) |
1.413 |
1.320 |
1.431 |
1.346 |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.362 |
0.327 |
0.377 |
0.381 |
n/a |
n/a |
n/a |
n/a |
SEMINAL VESICLE |
||||||||
- Absolute weight (g) |
0.843 |
0.816 |
0.827 |
0.805 |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.216 |
0.199 |
0.214 |
0.228 |
n/a |
n/a |
n/a |
n/a |
PROSTATE |
||||||||
- Absolute weight (g) |
1.002 |
0.957 |
0.969 |
0.776*** |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.256 |
0.236 |
0.254 |
0.219** |
n/a |
n/a |
n/a |
n/a |
BRAIN |
||||||||
- Absolute weight (g) |
2.071 |
2.069 |
2.015 |
2.026 |
1.883 |
1.946 |
1.961 |
nd |
- % body weight |
0.519 |
0.484 |
0.526 |
0.585* |
0.767 |
0.733 |
0.768 |
nd |
ADRENAL |
||||||||
- Absolute weight (g) |
0.070 |
0.069 |
0.070 |
0.076 |
0.086 |
0.076 |
0.093 |
nd |
- % body weight |
0.018 |
0.016 |
0.018 |
0.022 |
0.035 |
0.029* |
0.036 |
nd |
SPLEEN |
||||||||
- Absolute weight (g) |
0.605 |
0.651 |
0.554 |
0.526 |
0.577 |
0.504 |
0.491 |
nd |
- % body weight |
0.152 |
0.152 |
0.144 |
0.152 |
0.235 |
0.189** |
0.191** |
nd |
THYMUS |
||||||||
- Absolute weight (g) |
0.390 |
0.468 |
0.343 |
0.332 |
0.231 |
0.274 |
0.212 |
nd |
- % body weight |
0.098 |
0.109 |
0.090 |
0.096 |
0.093 |
0.104 |
0.082 |
nd |
LIVER |
||||||||
- Absolute weight (g) |
9.050 |
10.658** |
11.517** |
12.538** |
9.184 |
8.927 |
10.809 |
nd |
- % body weight |
2.272 |
2.489 |
2.993* |
3.594*** |
3.759 |
3.346 |
4.206 |
nd |
HEART |
||||||||
- Absolute weight (g) |
1.102 |
1.078 |
1.002 |
0.938*** |
0.859 |
0.798 |
0.849 |
nd |
- % body weight |
0.277 |
0.252 |
0.261 |
0.269 |
0.353 |
0.301 |
0.331 |
nd |
KIDNEY |
||||||||
- Absolute weight (g) |
2.257 |
2.473 |
2.717 |
2.426 |
1.841 |
1.747 |
1.864 |
nd |
- % body weight |
0.567 |
0.576 |
0.707* |
0.699* |
0.755 |
0.656 |
0.723 |
nd |
PITUITARY |
||||||||
- Absolute weight (g) |
0.010 |
0.009 |
0.010 |
0.009 |
0.012 |
0.012 |
0.013 |
nd |
- % body weight |
0.003 |
0.002 |
0.002 |
0.003 |
0.005 |
0.005 |
0.005 |
nd |
THYROID/ PARA |
||||||||
- Absolute weight (g) |
0.023 |
0.023 |
0.026 |
0.024 |
0.019 |
0.019 |
0.022 |
nd |
- % body weight |
0.006 |
0.006 |
0.007** |
0.007* |
0.008 |
0.007 |
0.009 |
nd |
OVARY |
||||||||
- Absolute weight (g) |
n/a |
n/a |
n/a |
n/a |
0.110 |
0.112 |
0.112 |
nd |
- % body weight |
n/a |
n/a |
n/a |
n/a |
0.044 |
0.044 |
0.044 |
nd |
UTERUS |
||||||||
- Absolute weight (g) |
n/a |
n/a |
n/a |
n/a |
0.501 |
0.457 |
0.496 |
nd |
- % body weight |
n/a |
n/a |
n/a |
n/a |
0.203 |
0.181 |
0.197 |
nd |
* P<0.05, ** P<0.01, ***p<0.001
nd: not determined
Table 8 Summary of mean estrous cycle data (n=10)
Predose |
Pre-pairing |
||||||||||||||
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
||||||||
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
2.3 |
4.5 |
2.5 |
4.1 |
2.1 |
4.9 |
2.2 |
4.4 |
2.4 |
4.5 |
2.2 |
4.1 |
2.4 |
4.2 |
2.2 |
5.0 |
Table 9 Summary of male fertility and reproductive performance
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Total males |
10 |
10 |
10 |
10 |
- Unscheduled deaths prior to cohabitation |
0 |
0 |
0 |
0 |
Males cohabitated |
10 |
10 |
10 |
10 |
- Unscheduled deaths during cohabitation |
0 |
0 |
0 |
1 |
Males mating with at least 1 female |
10 |
10 |
10 |
9 |
Males impregnating at least 1 female |
9 |
10 |
9 |
9 |
Mating index (%) |
100 |
100 |
100 |
90 |
Fecundity index (%) |
90 |
100 |
90 |
100 |
Fertility index (%) |
90 |
100 |
90 |
90 |
Mating index % = (no. of males mating with at least 1 female / no. of males cohabitated with at least 1 female) x 100
Fecundity index % = (no. of males impregnating at least 1 female / no. of males mating with at least 1 female) x 100
Fertility index % = (no. of males impregnating at least 1 female / no. of males cohabited with at least 1 female) x 100
Table 10 Summary of female fertility and reproductive performance
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Total females |
10 |
10 |
10 |
10 |
- Unscheduled deaths prior to cohabitation |
0 |
0 |
0 |
0 |
Females cohabitated |
10 |
10 |
10 |
10 |
- Unscheduled deaths during cohabitation |
0 |
0 |
0 |
0 |
Females mated |
10 |
10 |
10 |
10 |
Pregnant females |
9 |
10 |
9 |
10 |
- Pup(s) / total litter dead |
1 |
0 |
1 |
8 |
Non-pregnant females |
1 |
0 |
1 |
0 |
Mating per day periods of cohabitation: |
||||
- Day 1 |
2 |
4 |
1 |
1 |
- Day 2 |
2 |
0 |
2 |
1 |
- Day 3 |
4 |
2 |
3 |
3 |
- Day 4 |
2 |
4 |
2 |
3 |
- Day 7 |
0 |
0 |
1 |
2 |
- Day 12 |
0 |
0 |
1 |
0 |
Mating index % |
100 |
100 |
100 |
100 |
Fecundity index % |
90 |
100 |
90 |
100 |
Fertility index % |
90 |
100 |
90 |
100 |
Mating index % = (mated females / females cohabitated (exc. females sacrificed during cohabitation)) x 100
Fecundity index % = (pregnant females / mated females (exc. females with an undetermined pregnancy status)) x 100
Fertility index % = (pregnant females / females cohabitated (exc. females sacrificed during cohabitation or with an undetermined pregnancy status)) x 100
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- Amines, C12-14-branched alkyl, octylbenzenesulfonates (1:1)
- Molecular formula:
- Amines, C12-branched alkyl, octylbenzenesulfonates: C27H51NO3S Amines, C13-branched alkyl, octylbenzenesulfonates: C28H53NO3S Amines, C14-branched alkyl, octylbenzenesulfonates: C29H55NO3S
- IUPAC Name:
- Amines, C12-14-branched alkyl, octylbenzenesulfonates (1:1)
- Reference substance name:
- Amines, C12-14-branched alkyl, nonylbenzenesulfonates (1:1)
- Molecular formula:
- Amines, C12-branched alkyl, nonylbenzenesulfonates: C28H53NO3S Amines, C13-branched alkyl, nonylbenzenesulfonates: C29H55NO3S Amines, C14-branched alkyl, nonylbenzenesulfonates: C30H57NO3S
- IUPAC Name:
- Amines, C12-14-branched alkyl, nonylbenzenesulfonates (1:1)
- Reference substance name:
- Amines, C12-14-branched alkyl, decylbenzenesulfonates (1:1)
- Molecular formula:
- Amines, C12-branched alkyl, decylbenzenesulfonates: C29H55NO3S Amines, C13-branched alkyl, decylbenzenesulfonates: C30H57NO3S Amines, C14-branched alkyl, decylbenzenesulfonates: C31H59NO3S
- IUPAC Name:
- Amines, C12-14-branched alkyl, decylbenzenesulfonates (1:1)
- Reference substance name:
- Amines, C12-C14-branched alkyl, benzenesulfonates C12-C15 (1:1)
- Molecular formula:
- Amines, C12-branched alkyl, benzenesulfonates C12 (1:1) C31H59NO3S Amines, C12-branched alkyl, benzenesulfonates C13 (1:1) C32H61NO3S Amines, C12-branched alkyl, benzenesulfonates C14 (1:1) C33H63NO3S Amines, C12-branched alkyl, benzenesulfonates C15 (1:1) C34H65NO3S Amines, C13-branched alkyl, benzenesulfonates C12 (1:1) C32H61NO3S Amines, C13-branched alkyl, benzenesulfonates C13 (1:1) C33H63NO3S Amines, C13-branched alkyl, benzenesulfonates C14 (1:1) C34H65NO3S Amines, C13-branched alkyl, benzenesulfonates C15 (1:1) C35H67NO3S Amines, C14-branched alkyl, benzenesulfonates C12 (1:1) C33H63NO3S Amines, C14-branched alkyl, benzenesulfonates C13 (1:1) C34H65NO3S Amines, C14-branched alkyl, benzenesulfonates C14 (1:1) C35H67NO3S Amines, C14-branched alkyl, benzenesulfonates C15 (1:1) C36H69NO3S
- IUPAC Name:
- Amines, C12-C14-branched alkyl, benzenesulfonates C12-C15 (1:1)
- Reference substance name:
- Amines, C12-14-branched alkyl, hexadecylbenzenesulfonates (1:1)
- Molecular formula:
- Amines, C12-branched alkyl, hexadecylbenzenesulfonates (1:1): C35H67NO3S Amines, C13-branched alkyl, hexadecylbenzenesulfonates (1:1): C36H69NO3S Amines, C14-branched alkyl, hexadecylbenzenesulfonates (1:1): C37H71NO3S
- IUPAC Name:
- Amines, C12-14-branched alkyl, hexadecylbenzenesulfonates (1:1)
- Reference substance name:
- Residual Amines, C12-14-tert-alkyl
- IUPAC Name:
- Residual Amines, C12-14-tert-alkyl
- Test material form:
- liquid: viscous
- Details on test material:
- Storage: Room Temperature
Constituent 1
Constituent 2
Constituent 3
Constituent 4
Constituent 5
Constituent 6
Test animals
- Species:
- rat
- Strain:
- other: CRL:WI(Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported, assume yes
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: Males: 288.6 - 391.8 g; females: 173.2 - 235.3 g
- Fasting period before study: No
- Housing: During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation, and with their litter during the lactation phase. Males were returned to group-housing after the pairing phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum): Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum): Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
- Acclimation period: Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for at least 14 days prior to initiation of dosing (males) or 7 days prior to initiation of smearing (females).
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12:12
IN-LIFE DATES: From: 26 July 2017 To: 25 September 2017
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- - PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly. The test article was formulated as a suspension in Corn Oil. Formulations were stored at room temperature (15 to 25C) in a sealed container, protected from the light.
- VEHICLE
- Justification for use and choice of vehicle (if other than water): Homogenous stable solutions were prepared in corn oil, a guideline recommended vehicle.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): MKBP7039V, MKBZ9899V and MKCC0462
- Purity: Assumed pure corn oil - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Single 5.0 mL aliquot formulation samples were collected from the main in-life experiment study for each animal group at the respective concentrations (0, 20.0, 60.0 and 100.0 mg/mL) on the 26th July 2017 (week 1 of the in-life phase), 9th August 2017 (week 3 of the in-life phase) and 30th August 2017 (week 6 of the in-life phase) for analysis.
A validated gradient elution reversed-phase high-performance liquid chromatographic method with ultraviolet detection (HPLC-UV) was employed for determination of formulation stability and homogeneity, and concentration analyses of formulations administered during the study.
No test item was observed in the control group samples. Measured concentrations from the test item groups were found to be in the range of 89.2 - 102.3 % of nominal values. - Duration of treatment / exposure:
- Males: 42 consecutive days (2 weeks prior to pairing; during pairing; and until the day before necropsy)
Females: 61 days (2 weeks prior to pairing [pre-pairing phase]; during the pairing phase; and until LD 13, inclusive, or 25 days post-coitum for females which did not litter and were sent to necropsy on LD 14 or 26 days post coitum). Some females were not dosed on LD 0 if they were observed to be starting or just completed parturition. - Frequency of treatment:
- Daily
Doses / concentrationsopen allclose all
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10 males and 10 females per treatment group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses selected based on the results of a preliminary range-finder test.
- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A - Positive control:
- N/A
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
BODY WEIGHT: Yes
- Time schedule: Male body weights were recorded once during acclimation, before dosing on the first day of dosing, at weekly intervals, and before necropsy. Female body weights were recorded once during acclimation; before dosing on the first day of dosing; at weekly intervals prior to pairing and until confirmation of mating; on Gestation Day (GD) 0, 7, 14, and 20; and on LD 1, 4, 13 and 14 (prior to necropsy).
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g/animal/day: Yes
- The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and LD 0 to 13. Consumption was calculated as g/animal/day.
WATER CONSUMPTION: Yes
- Time schedule: The amount of water consumed was determined daily during the pre-pairing phase (both sexes). Consumption was calculated as g/animal/day.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes (identity); isoflurane
- Animals fasted: Yes
- How many animals: All
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Animals fasted: Yes
- How many animals: All
URINALYSIS: Yes
- Time schedule for collection of urine: Collected overnight during Week 6
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity / alertness / approach response / acoustic startle response / bar test / extensor thrust / pain response / righting reflex
IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.
OTHER:
Locomotor activity was assessed in an automated photocell activity recorder for 30 minutes and was undertaken for five selected animals/sex/group (five males with the highest identification numbers and the first five littered females/group). Assessments were performed during Week 6 of dosing (Post Pairing Day 8) for males and on LD 7 for females.
Activity counts were recorded at 5-minute intervals. The following parameters were determined; total activity / total mobile counts
Five selected animals/sex/group (five males with the highest identification numbers and the first five littered females/group) were observed in the hand and in an arena. Assessments were performed during Week 6 of dosing (Post Pairing Day 12) for males and on LD 7 for females. Quantitative assessment parameters are as follows: hind limb foot splay / fore and/or hind limb grip strength - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Statistics:
- Data for each sex were analyzed separately, unless stated otherwise. Only data collected on or after the first day of dosing were analyzed statistically. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P ≤ 0.05. By default, significant results were reported as *P ≤ 0.05, +P ≤ 0.01, and/or #P ≤ 0.001.
See details in attached study report.
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Urogenital staining and wet abdominal fur were occasionally recorded during Pre Pairing and Pairing for one or two males administered 300 or 500 mg/kg/day. An observation of vocalizing on handling was noted in one female from each test article treated group from Pre-Pairing Day 4 or 5. One female administered 500 mg/kg/day was noted to have wet fur of the anogenital area and/or abdomen during the pre-pairing phase or GD 7. These observations were attributed to test article.
Incidences of mouth rubbing were noted immediately upon the return to the home cage in up to 4 of 10 females administered 500 mg/kg/day. Salivation was observed from Pre-Pairing Day 8 for some females administered 100, 300, or 500 mg/kg/day and persisted through pairing for occasional females, but no dose response was noted. Salivation was not recorded during gestation or lactation. Salivation was also noted for males administered 100, 300, or 500 mg/kg/day, without a dose response, from Pre-Pairing Day 9, 7, and 10, respectively, and until the end of the pre-pairing, through the pairing, and into the post pairing phases. These observations were attributed to the consistency and palatability of the test article and were considered non-adverse.
All other observations, including fur staining, fur loss, sores/lesions, and tail damage, were considered incidental and not related to the test article as they were also observed in control animals, showed no dose relationship, and/or are commonly observed in animals of this age and strain. - Mortality:
- not specified
- Description (incidence):
- One male administered 500 mg/kg/day was found dead on Study Day 16 (Pairing Day 1). No adverse clinical observation were noted prior to the animal being found dead, and no specific cause of demise could be determined from the tissues examined. The death, therefore, had an uncertain relationship to the test article.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Test article-related lower mean body weight gain was recorded for males administered 300 or 500 mg/kg/day. Mean male body weight gain was statistically significantly reduced during the pre pairing phase for males administered 500 mg/kg/day and was 67% lower than controls by the end of the pre-pairing phase. Thereafter, mean body weight gain for males was similar to or slightly lower than controls, but this did not attain statistical significance. Overall, the mean male body weight gain at completion of the study was statistically significantly reduced at 57% of the control value for males administered 500 mg/kg/day. While individual incidences did not attain statistical significance for males administered 300 mg/kg/day, the overall gain for the study showed a statistically significantly reduction (77% of the control value). Mean body weight was unaffected in males administered 100 mg/kg/day, compared with controls.
Body weight gain was also lower for females administered 500 mg/kg/day, when compared with controls during late gestation.
Mean body weight during the pre-pairing, gestation, or lactation phases for females administered 100, 300 mg/kg/day were compared with controls and considered unaffected by the test article. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Lower food consumption was noted for males and females administrated 500 mg/kg/day during the first two weeks of dosing. Food consumption was also noted to be lower for 500 mg/kg/day females during late gestation, when compared with controls.
No effect on food consumption was noted following 100 or 300 mg/kg/day administration. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- A test article related effect on water consumption was noted for animals administered 300 or 500 mg/kg/day.
Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, compared with controls, such that at the end of the pre-pairing phase, male water consumption was 67 or 100% greater than controls, respectively, and female water consumption was 38 or 80% greater than controls, respectively. - Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Hematology parameters were unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- A statistically significant elevation in glucose levels was noted in males administered 500 mg/kg/day, and a statistically significant increase in cholesterol, calcium, and inorganic phosphate levels was noted in males administered 300 or 500 mg/kg/day, compared with control. Cholesterol and glucose levels were also elevated in females administered 300 mg/kg/day.
A statistically significant increase in TSH was noted in males administered 300 mg/kg/day (155 % of control) and animals administered 500 mg/kg/day (188 and 176 % of control) following approximately 6 weeks of dosing. These values fell outside of background control ranges. - Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean urine volume was increased for males administered 300 or 500 mg/kg/day, compared with controls (121 and 86 % of control values, respectively); the increase attained statistical significance for males administered 300 mg/kg/day.
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- During the assessments, moderate salivation, piloerection, and changes in body/head posture were observed for males in all test article treated groups, compared with controls. In addition, a dose related increase in ataxia was noted, and high stepping, walking on toes, and slow deliberate movements were noted for animals administered 300 or 500 mg/kg/day, although a decrease in activity was not observed in these groups, compared with controls. A statistically significant increased number of rears were noted on Pre-Pairing Day 6 for males administered 300 or 500 mg/kg/day and Post-Pairing Day 12 for males administered 500 mg/kg/day, compared with controls.
Female changes during the assessments included piloerection and salivation, which were generally dose related and behavior changes predominantly characterized by paw flicking (fore paws). A statistically significant decreased number of rears were noted on LD 1 and 7 for females 300 mg/kg/day, compared with controls. A lower number was also recorded on LD 1 for females administered 500 mg/kg/day; these females were early decedents due to litter loss and, therefore, no data are available for LD 7. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Group mean adjusted liver weights were increased for males administered 300 or 500 mg/kg/day, compared with controls, which generally correlated macroscopically with mottled and/or pale discoloration and/or with the microscopic finding of centrilobular hepatocyte hypertrophy.
Group mean adjusted thyroid weights were increased for males administered 300 or 500 mg/kg/day, compared with controls, which generally correlated macroscopically with dark discoloration and/or with the microscopic finding of follicular cell hypertrophy.
Group mean adjusted kidney weights were increased for males administered 300 or 500 mg/kg/day, compared with controls, which generally correlated macroscopically with mottled and/or pale discoloration and/or with the microscopic finding of nephropathy.
All other organ weight changes, including those statistically significant, were attributed to normal biological variation and were considered not test article related as they were small in magnitude, not dose-dependent, inconsistent between sexes, due to normal inter-animal variability, and/or lacked a microscopic correlate. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In the liver, mottled and/or pale discoloration were recorded in four males administered 100 mg/kg/day, five males administered 300 mg/kg/day and four males administered 500 mg/kg/day.
In the thyroid, dark discoloration was recorded in one male administered 500 mg/kg/day.
In the kidney, mottled and/or pale were recorded in three males administered 100 mg/kg/day, six males administered 300 mg/kg/day and four males administered 500 mg/kg/day.
In the stomach, mucosal thickening was recorded in two males administered 300 mg/kg/day and one male administered 500 mg/kg/day. Raised focus was recorded in one male administered 500 mg/kg/day.
Small thymus was recorded in one female administered 300 mg/kg/day. - Neuropathological findings:
- no effects observed
- Description (incidence and severity):
- Locomotor activity was unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- A range of Amines-related findings were recorded in the liver, thyroid, kidney, and nonglandular stomach of animals administered 100, 300, or 500 mg/kg/day.
In the liver, centrilobular hepatocyte hypertrophy was recorded in two males administered 100 mg/kg/day, all males and all examined females administered 300 or 500 mg/kg/day. This finding was characterized by the enlargement of the hepatocytes, a ground glass appearance of the cytoplasm, and slight compression of the adjacent sinusoids; was principally localized within the centrilobular areas, but occasionally also extended more diffusely to midzonal and periportal regions in affected animals; and correlated generally with increased group mean adjusted liver weights and the macroscopic finding of dark/mottled discoloration.
In the thyroid, follicular cell hypertrophy was recorded in one male administered 100 mg/kg/day, most males and four females administered 300 mg/kg/day and all males and one female administered 500 mg/kg/day and was characterized by follicular cells with increased amounts of cytoplasm, follicular epithelium that ranged from flat to cuboidal or columnar and follicles with decreased amounts of colloid. This finding correlated generally with increased group mean adjusted thyroid weights and with the macroscopic finding of dark discoloration in one male administered 500 mg/kg/day.
In the kidney, nephropathy was recorded in all examined males administered Amines, which was characterized by multifocal areas of tubular basophilia, inflammatory foci dominated by lymphocytes and/or plasma cells, and granular casts principally located at the corticomedullary junction, but occasionally also in cortical and/or papillary tubules. In addition, hyaline droplets in the cytoplasm of tubular epithelial cells of males administered 100, 300 or 500 mg/kg/day were recorded with higher gradings, compared with concurrent controls. This finding was characterized by focally extensive areas of brightly eosinophilic aggregates of cytoplasmic droplets principally located in the superficial cortex, but multifocally extending towards to the corticomedullary junction. Both of these microscopic findings correlated generally with increased group mean adjusted kidney weights and with the macroscopic finding of mottled and/or pale discoloration.
In addition, in the nonglandular stomach, epithelial hyperplasia and orthokeratotic hyperkeratosis were recorded in one male administered 300 mg/kg/day and four males administered 500 mg/kg/day; these correlated generally with mucosal thickening or the presence of a raised focus, macroscopically - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not examined
Effect levels
open allclose all
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- behaviour (functional findings)
- body weight and weight gain
- clinical biochemistry
- clinical signs
- gross pathology
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- urinalysis
- water consumption and compound intake
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- clinical signs
- histopathology: non-neoplastic
- water consumption and compound intake
Target system / organ toxicity
- Key result
- Critical effects observed:
- no
Any other information on results incl. tables
Table 4 Group mean achieved dose rates for each treatment group (in mg/kg bw/day)
Test Group (mg/kg/day) |
Nominal formulation concentration (mg/mL) |
Actual formulation concentration achieved (% of nominal) |
||
Week 1 |
Week 3 |
Week 6 |
||
Control |
0 |
nd |
nd |
nd |
100 |
20 |
95.8 |
97.3 |
102.3 |
300 |
60 |
96.9 |
95.7 |
96.3 |
500 |
100 |
94.8 |
95.9 |
89.2 |
nd: not detected
Table 5 Mean body weights and body weight gains/ losses (n=10 unless stipulated otherwise) (F0)
Sex / Day |
Bodyweight (g) |
|||
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
|
MALES |
||||
Predose |
||||
Day 1 |
301.5 |
317.7 |
313.0 |
305.7 |
Pre-pairing |
||||
Day 1 |
325.1 |
342.6 |
339.0 |
328.4 |
Day 8 |
345.1 |
365.8 |
355.2 |
331.6 |
Day 15 |
363.1 |
382.3 |
370.8 |
341.0 |
Change Day 1-8 |
20.1 |
23.2 |
16.2 |
3.3** |
Change Day 8-15 |
18.0 |
16.5 |
15.5 |
9.3* |
Pairing |
||||
Day 7 |
369.2 |
389.2 |
372.7 |
348.6 (n=9) |
Day 14 |
385.1 |
406.3 |
385.5 |
365.1 (n=9) |
Change Day 7-14 |
15.9 |
17.1 |
12.8 |
16.5 |
Post-pairing |
||||
Day 7 |
400.6 |
420.8 |
396.2 |
374.9 (n=9) |
Day 13 |
407.5 |
424.2 |
402.2 |
378.2 (n=9) |
Change Day 7-13 |
6.8 |
3.4 |
6.0 |
3.3 |
Overall weight change (pre-pairing Day 1 – post-pairing Day 13) |
82.5 |
81.6 |
63.2* |
47.0** |
FEMALES |
||||
Predose |
||||
Day 8 |
190.1 |
189.6 |
208.6 |
217.3 |
Pre-pairing |
||||
Day 1 |
198.1 |
197.9 |
208.6 |
215.2 |
Day 8 |
208.6 |
199.4 |
212.1 |
225.4 |
Day 15 |
217.3 |
200.6 |
209.4 |
219.4 |
Change Day 1-8 |
10.5 |
10.8 |
12.8 |
8.8 |
Change Day 8-15 |
8.6 |
6.5 |
13.3 |
10.0 |
Gestation |
||||
Day 0 |
220.6 (n=9) |
242.0 |
268.4 (n=9) |
326.9 (n=9) |
Day 7 |
217.5 (n=9) |
238.8 |
263.4 (n=9) |
314.7 (n=9) |
Day 14 |
228.0 (n=9) |
247.3 |
277.1 (n=9) |
332.3 (n=9) |
Day 20 |
221.0 (n=9) |
244.6 |
269.1 (n=9) |
311.6 (n=9) |
Change Day 0-7 |
21.4 |
21.3 |
19.4 |
23.7 |
Change Day 7-14 |
26.4 |
24.5 |
29.8 |
24.4 |
Change Day 14-20 |
58.5 |
51.3 |
55.2 |
42.6 |
Lactation |
||||
Day 1 |
252.8 (n=9) |
252.3 |
255.3 (n=8) |
- |
Day 4 |
256.6 (n=8) |
259.4 |
261.6 (n=8) |
- |
Day 13 |
275.5 (n=8) |
274.9 |
286.8 (n=8) |
- |
Day 14 |
258.2 (n=8) |
255.0 |
254.9 (n=8) |
- |
Change Day 1-4 |
3.7 |
7.1 |
6.4 |
- |
Change Day 4-13 |
18.9 |
15.6 |
25.2 |
- |
Change Day 13-14 |
-17.3 |
-20.0 |
-31.9* |
- |
Overall weight change (pre-pairing Day 1 – gestation Day 20) |
128.6 |
116.8 |
134.0 |
110.7 |
* p<0.05, **p<0.01
Table 6 Haematology, clinical chemistry, urinalysis and pathology findings (F0)
Doses (ppm) |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Male |
Female |
|||||||
Haematology |
||||||||
Haemoglobin (g/dl) |
14.6 |
15.2 |
15.1 |
14.7 |
14.8 |
14.7 |
14.8 |
15.5 # |
Red blood cells (1012/L) |
8.34 |
8.56 |
8.48 |
8.22 |
7.77 |
7.70 |
7.64 |
7.93 # |
Packed cell volume (%) |
45.4 |
46.9 |
47.2 |
45.5 |
44.1 |
44.7 |
44.9 |
48.5 # |
Mean cell volume (fL) |
54.5 |
54.9 |
55.7 |
55.3 |
56.8 |
58.0 |
58.9 |
61.1 # |
Mean cell haemoglobin (pg) |
17.5 |
17.7 |
17.9 |
17.8 |
19.0 |
19.1 |
19.4 |
19.6 # |
Mean cell haemoglobin conc. (g/dL) |
32.2 |
32.3 |
32.1 |
32.3 |
33.4 |
33.0 |
33.0 |
32.1 # |
Reticulocytes (%) |
2.5 |
2.2 |
2.5 |
2.4 |
3.4 |
3.8 |
3.7 |
2.8 # |
Absolute reticulocytes (109/L) |
204.3 |
189.3 |
208.6 |
197.2 |
261.9 |
294.6 |
284.5 |
218.6 # |
Red blood cell distribution (%) |
12.5 |
13.1 |
13.3 |
13.6 |
12.5 |
12.4 |
14.5 |
14.2 # |
Haemoglobin distr. width (g/dL) |
2.32 |
2.28 |
2.29 |
2.28 |
1.93 |
1.91 |
1.95 |
1.76 # |
White blood cells (109/L) |
3.6 |
3.8 |
4.6 |
4.6 |
5.2 |
4.9 |
4.3 |
3.0 # |
Neutrophils (109/L) |
0.75 |
0.66 |
0.86 |
0.77 |
2.84 |
2.15 |
1.81 |
1.40 # |
Lymphocytes (109/L) |
2.69 |
2.98 |
3.56 |
3.62 |
2.11 |
2.46 |
2.20 |
1.47 # |
Monocytes (109/L) |
0.05 |
0.06 |
0.09** |
0.08 |
0.17 |
0.17 |
0.22 |
0.11 # |
Eosinpphils (109/L) |
0.05 |
0.06 |
0.06 |
0.05 |
0.05 |
0.04 |
0.04 |
0.04 # |
Basophils (109/L) |
0.00 |
0.01 |
0.01 |
0.01 |
0.00 |
0.00 |
0.01 |
0.00 # |
Large unstained cells (109/L) |
0.01 |
0.01 |
0.03 |
0.02 |
0.04 |
0.0 |
0.06 |
0.01 # |
Neutrophils (%) |
24 |
20 |
19 |
17 |
56 |
43 |
41 |
46 # |
Lymphocytes (%) |
73 |
76 |
77 |
80 |
39 |
53 |
52 |
49 # |
Monocytes (%) |
2 |
2 |
2 |
2 |
4 |
3 |
5 |
4 # |
Eosinophils (%) |
2 |
2 |
2 |
1 |
1 |
1 |
1 |
1 |
Basophils (%) |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Large unstained cells (%) |
0 |
0 |
1 |
0 |
1 |
1 |
1 |
0 |
Platelets (109/L) |
729 |
728 |
771 |
782 |
936 |
932 |
895 |
636 # |
Platelet crit (%) |
0.58 |
0.59 |
0.61 |
0.62 |
0.61 |
0.63 |
0.60 |
0.40 # |
Mean platelet volume (fL) |
8.1 |
8.1 |
7.8 |
7.9 |
6.5 |
6.8 |
6.7 |
6.3 # |
Platelet distribution width (%) |
55.5 |
55.7 |
52.4 |
54.4 |
51.9 |
50.1 |
49.9 |
50.2 # |
Prothrombin time (sec) |
20.1 |
20.9 |
21.1 |
21.1 |
22.2 |
22.5 |
23.7 |
21.5 # |
Thromboplastin time(sec) |
16.7 |
17.2 |
17.5 |
16.8 |
24.0 |
19.8 |
18.3 |
15.7 # |
Fibrinogen (g/L) |
1.34 |
1.37 |
1.42 |
1.42 |
2.11 |
1.93 |
2.07 |
1.70 # |
Blood chemistry |
||||||||
Aspartate aminotransferase (IU/L) |
56 |
47** |
51 |
46* |
111 |
94 |
104 |
62 # |
Alanine aminotransferase (IU/L) |
38 |
30 |
39 |
42 |
65 |
61 |
75 |
45 # |
Alkaline phosphatase (IU/L) |
70 |
77 |
84 |
94 |
74 |
65 |
77 |
98 # |
Total cholesterol (mmol/L) |
1.5 |
1.7 |
1.9* |
2.0** |
2.1 |
2.1 |
2.6* |
2.5 # |
Total bilirubin (µmol/L) |
<1.7 |
<1.7 |
<1.7 |
<1.7 |
<1.7 |
<1.7 |
<1.7 |
<1.7 |
Total protein (g/L) |
59 |
59 |
59 |
57 |
56 |
56 |
59 |
58 # |
Albumin (g/L) |
39 |
39 |
37 |
37 |
32 |
33 |
35 |
35 # |
Globulin (g/L) |
20 |
21 |
22 |
20 |
25 |
23 |
25 |
23 # |
Albumin/Globulin ratio |
1.9 |
1.9 |
1.7 |
1.9 |
1.3 |
1.5 |
1.4 |
1.5 # |
Sodium (mmol/L) |
142 |
142 |
142 |
141 |
138 |
13 |
138 |
140 # |
Potassium (mmol/L) |
3.7 |
3.6 |
3.8 |
3.7 |
3.6 |
3.5 |
3.8 |
3.8 # |
Chloride (mmol/L) |
102 |
102 |
102 |
102 |
95 |
96 |
98 |
99 # |
Calcium (mmol/L) |
2.49 |
2.54 |
2.57* |
2.58* |
2.69 |
2.73 |
2.75 |
2.78 # |
Inorganic phosphate (mmol/L) |
1.6 |
1.6 |
1.9* |
1.9* |
3.3 |
3.4 |
3.2 |
3.0 # |
Enzymatic creatinine (µmol/L) |
27 |
31 |
29 |
30 |
46 |
46 |
38 |
32 # |
Urea (mmol/L) |
5.7 |
5.9 |
5.5 |
6.2 |
16.6 |
15.6 |
14.5 |
10.4 # |
Glucose (mmol/L) |
8.8 |
9.9 |
10.1 |
11.0** |
8.4 |
8.9 |
10.2* |
7.9 # |
Serum bile acids (µmol/L) |
14.40 |
<14.45 |
<15.66 |
15.22 |
38.89 |
22.33 |
50.98 |
12.08 # |
Urinalysis |
||||||||
Volume (mL) |
12.6 |
11.5 |
27.8* |
23.4 |
- |
- |
- |
- |
Specific gravity (g/L) |
1.020 |
1.019 |
1.011 |
1.015 |
- |
- |
- |
- |
Thyroid hormone analysis |
||||||||
Total T3 (nmol/L) |
<0.57 |
<0.57 |
<0.54 |
<0.54 |
- |
- |
- |
- |
Imulite Total T4 (nmol/L) |
59 |
55 |
57 |
48 |
- |
- |
- |
- |
Rapid thyroid stim. hormone (µlU/mL) |
0.33 |
0.42 |
0.84* |
0.95** |
- |
- |
- |
- |
Pathology (remarkable changes) |
||||||||
Adrenals - pale, moderate - large, slight - pale, slight |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=5) 1 0 0 |
(n=5) 0 1 1 |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=1) 0 0 0 |
Thyroid - dark, slight - pale, slight - small, slight |
(n=10) 0 0 0 |
(n=10) 0 0 0 |
(n=10) 0 0 0 |
(n=9) 1 0 0 |
(n=8) 0 1 1 |
(n=10) 0 0 0 |
(n=8) 0 2 1 |
(n=1) 0 0 0 |
Thymus - dark, minimal - red focus - small, slight |
(n=5) 0 0 0 |
(n=5) 1 0 0 |
(n=5) 0 1 0 |
(n=5) 0 1 0 |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=5) 0 0 1 |
(n=1) 0 0 0 |
Lymph node, mesenteric - red, slight |
(n=5) 0 |
(n=5) 2 |
(n=5) 0 |
(n=5) 1 |
(n=5) 1 |
(n=5) 1 |
(n=5) 1 |
(n=1) 0 |
Liver - mottled, moderate - mottled, slight - pale, moderate - pale, slight |
(n=7) 1 1 0 2 |
(n=5) 0 4 0 1 |
(n=7) 0 3 1 2 |
(n=7) 1 3 0 2 |
(n=5) 0 0 0 0 |
(n=5) 0 0 0 0 |
(n=5) 0 0 0 0 |
(n=1) 0 0 0 0 |
Stomach - raised focus, pale - red, slight - striation, red - thick, minimal - thick, slight |
(n=5) 0 1 0 0 0 |
(n=5) 0 1 0 0 0 |
(n=5) 0 1 2 1 1 |
(n=5) 1 0 2 0 1 |
(n=5) 0 0 0 0 0 |
(n=5) 0 1 0 0 0 |
(n=5) 0 0 0 0 0 |
(n=1) 0 0 0 0 0 |
Colon - distension, slight |
(n=5) 0 |
(n=5) 0 |
(n=5) 1 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=1) 0 |
Lymph node, mandibular - red, moderate |
(n=5) 0 |
(n=5) 1 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=1) 0 |
Kidney - mottled, minimal - mottled, moderate - mottled, slight - pale, moderate - pale, slight - pelvic dilation, minimal - pelvic dilation, slight - red area |
(n=5) 0 0 0 0 0 0 0 0 |
(n=5) 0 0 2 0 2 0 0 1 |
(n=8) 0 1 5 1 0 1 1 0 |
(n=7) 1 1 2 1 0 0 0 0 |
(n=5) 0 0 0 0 0 0 0 0 |
(n=5) 0 0 0 0 0 0 0 0 |
(n=5) 0 0 0 0 0 0 1 0 |
(n=5) 0 0 0 0 0 0 0 0 |
Testis - small, severe |
(n=10) 0 |
(n=10) 1 |
(n=10) 0 |
(n=9) 0 |
n/a |
n/a |
n/a |
n/a |
Epididymis - small, marked |
(n=10) 0 |
(n=10) 1 |
(n=10) 0 |
(n=9) 0 |
n/a |
n/a |
n/a |
n/a |
Skin - fur loss, moderate - fur loss, slight - sore, slight |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=2) 1 0 1 |
(n=2) 0 2 1 |
(n=0) 0 0 0 |
Lung - pale focus |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 1 |
(n=1) 0 |
Tail - kinked |
(n=0) 0 |
(n=0) 0 |
(n=0) 0 |
(n=0) 0 |
(n=2) 2 |
(n=0) 0 |
(n=0) 0 |
(n=0) 0 |
Peyer’s Patch - prominent, moderate |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 1 |
(n=5) 0 |
(n=5) 0 |
(n=1) 0 |
Mammary gland - thick, moderate |
(n=10) 0 |
(n=10) 0 |
(n=10) 0 |
(n=9) 0 |
(n=8) 0 |
(n=10) 1 |
(n=8) 0 |
(n=1) 0 |
* P<0.05, ** P<0.01
# excluded from dataset
Table 7 Absolute and adjusted organ weights (F0)
Doses (mg/kg/day) |
Control |
100 |
300 |
500 |
Control |
100 |
300 |
500 |
MALE |
FEMALE |
|||||||
BODY WEIGHT |
||||||||
Weight (g) |
391.8 |
407.9 |
381.0 |
356.0* |
249.8 |
253.8 |
252.2 |
|
TESTES |
||||||||
- Absolute weight (g) |
3.561 |
3.534 |
3.724 |
3.781 |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.912 |
0.878 |
0.982 |
1.068 |
n/a |
n/a |
n/a |
n/a |
EPIDIDYMIS |
||||||||
- Absolute weight (g) |
1.413 |
1.320 |
1.431 |
1.346 |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.362 |
0.327 |
0.377 |
0.381 |
n/a |
n/a |
n/a |
n/a |
SEMINAL VESICLE |
||||||||
- Absolute weight (g) |
0.843 |
0.816 |
0.827 |
0.805 |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.216 |
0.199 |
0.214 |
0.228 |
n/a |
n/a |
n/a |
n/a |
PROSTATE |
||||||||
- Absolute weight (g) |
1.002 |
0.957 |
0.969 |
0.776*** |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.256 |
0.236 |
0.254 |
0.219** |
n/a |
n/a |
n/a |
n/a |
BRAIN |
||||||||
- Absolute weight (g) |
2.071 |
2.069 |
2.015 |
2.026 |
1.883 |
1.946 |
1.961 |
nd |
- % body weight |
0.519 |
0.484 |
0.526 |
0.585* |
0.767 |
0.733 |
0.768 |
nd |
ADRENAL |
||||||||
- Absolute weight (g) |
0.070 |
0.069 |
0.070 |
0.076 |
0.086 |
0.076 |
0.093 |
nd |
- % body weight |
0.018 |
0.016 |
0.018 |
0.022 |
0.035 |
0.029* |
0.036 |
nd |
SPLEEN |
||||||||
- Absolute weight (g) |
0.605 |
0.651 |
0.554 |
0.526 |
0.577 |
0.504 |
0.491 |
nd |
- % body weight |
0.152 |
0.152 |
0.144 |
0.152 |
0.235 |
0.189** |
0.191** |
nd |
THYMUS |
||||||||
- Absolute weight (g) |
0.390 |
0.468 |
0.343 |
0.332 |
0.231 |
0.274 |
0.212 |
nd |
- % body weight |
0.098 |
0.109 |
0.090 |
0.096 |
0.093 |
0.104 |
0.082 |
nd |
LIVER |
||||||||
- Absolute weight (g) |
9.050 |
10.658** |
11.517** |
12.538** |
9.184 |
8.927 |
10.809 |
nd |
- % body weight |
2.272 |
2.489 |
2.993* |
3.594*** |
3.759 |
3.346 |
4.206 |
nd |
HEART |
||||||||
- Absolute weight (g) |
1.102 |
1.078 |
1.002 |
0.938*** |
0.859 |
0.798 |
0.849 |
nd |
- % body weight |
0.277 |
0.252 |
0.261 |
0.269 |
0.353 |
0.301 |
0.331 |
nd |
KIDNEY |
||||||||
- Absolute weight (g) |
2.257 |
2.473 |
2.717 |
2.426 |
1.841 |
1.747 |
1.864 |
nd |
- % body weight |
0.567 |
0.576 |
0.707* |
0.699* |
0.755 |
0.656 |
0.723 |
nd |
PITUITARY |
||||||||
- Absolute weight (g) |
0.010 |
0.009 |
0.010 |
0.009 |
0.012 |
0.012 |
0.013 |
nd |
- % body weight |
0.003 |
0.002 |
0.002 |
0.003 |
0.005 |
0.005 |
0.005 |
nd |
THYROID/ PARA |
||||||||
- Absolute weight (g) |
0.023 |
0.023 |
0.026 |
0.024 |
0.019 |
0.019 |
0.022 |
nd |
- % body weight |
0.006 |
0.006 |
0.007** |
0.007* |
0.008 |
0.007 |
0.009 |
nd |
OVARY |
||||||||
- Absolute weight (g) |
n/a |
n/a |
n/a |
n/a |
0.110 |
0.112 |
0.112 |
nd |
- % body weight |
n/a |
n/a |
n/a |
n/a |
0.044 |
0.044 |
0.044 |
nd |
UTERUS |
||||||||
- Absolute weight (g) |
n/a |
n/a |
n/a |
n/a |
0.501 |
0.457 |
0.496 |
nd |
- % body weight |
n/a |
n/a |
n/a |
n/a |
0.203 |
0.181 |
0.197 |
nd |
* P<0.05, ** P<0.01, ***p<0.001
nd: not determined
Applicant's summary and conclusion
- Conclusions:
- Test article-related findings observed in males following 100 mg/kg/day administration were generally confined to microscopic changes in the liver and thyroid, and a rat specific change in the kidneys. These findings were considered not to represent an adverse health effect in humans, and hence, the no observed adverse effect level (NOAEL) for male systemic toxicity was established as 100 mg/kg/day.
No test article-related changes were noted for maternal females or offspring following administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for maternal systemic toxicity, and the subsequent progeny, was established as 100 mg/kg/day. - Executive summary:
OECD 422 (2018) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Amines, C12-12-branched alkyl, docdecylbenzenesulfonates (1:1) was administered to 30 male and 30 female Crl:WI(Han) strain rats by oral gavage administration at dose levels of 100, 300 and 500 mg/kg/day for up to 6 weeks. In addition, 10 male and 10 female were used as control rats and were treated with the corn oil vehicle only.
A summary of adult responses to the test item are described below;
Males
One male administered 500 mg/kg/day was found dead on Study Day 16. No adverse clinical observations were noted, and no specific cause of demise could be determined macroscopically or microscopically. The cause of death in relation to the test article could not be excluded.
Urogenital staining and wet abdominal fur was recorded occasionally for males administered 300 or 500 mg/kg/day. During the FOB assessment, salivation; piloerection; changes in body posture; and a dose‑related increase in ataxia, high stepping, walking on toes, and slow deliberate movements were noted. Additionally, increased incidences of rears were noted for males administered 300 or 500 mg/kg/day, compared with controls. These observations were considered an adverse test article-related effect.
The most marked reduction in body weight gain was noted during the pre‑pairing phase, with a statistically significant reduction noted for males administered 500 mg/kg/day. However, reduction in body weight gain continued to be noted, and the mean body weight gain after 6 weeks of dosing was statically significantly reduced for males administered 300 or 500 mg/kg/day, compared with controls. This reduction in body weight gain was not associated with a reduction in food consumption.
Clinical pathology parameters affected include increased glucose, cholesterol, calcium, inorganic phosphate, and increased urine output for animals administered 300 or 500 mg/kg/day. Mean water consumption was also increased, but as water consumption values were only collected for the first 2 weeks of study, this increase suggested test article-related kidney damage. This was also supported by microscopic observations noted; nephropathy and hyaline droplet accumulation in the renal tubules. These observations may indicate enhanced or accelerated development of glomerulosclerosis.
The epidermal hyperplasia and/or hyperkeratosis in the nonglandular stomach were considered consistent with a chronic irritation-type reaction.
Hepatocyte hypertrophy in the liver and the associated increase in TSH and thyroid follicular cell hypertrophy is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites and is considered rat specific.
Females
In general, females were less affected than males. No test article-related effects on clinical or postdose observations were noted, although lower body weight gain and food consumption was noted during late gestation following 500 mg/kg/day administration.
Some markers of toxicity were observed. The FOB observations included piloerection, salivation, and paw flicking (fore paws), which increased in a dose‑dependent manner. A decreased number of rears was also noted during the lactation phase for females 300 mg/kg/day, compared with controls. Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, at 38 or 80 % greater than the controls, respectively. Cholesterol and glucose levels were also elevated in females administered 300 mg/kg/day. However, the macroscopic and/or microscopic observations noted in the kidney of males were not apparent in females.
Microscopic observations included epidermal hyperplasia and/or hyperkeratosis in the non-glandular stomach, which were considered consistent with a chronic irritation-type reaction.
Hepatocyte hypertrophy in the liver and the associated increase in TSH and thyroid follicular cell hypertrophy were less pronounced in females than males; nevertheless, it is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites and is considered rat specific.
Endpoint Discussion
Test article-related findings observed in males following 100 mg/kg/day administration were generally confined to microscopic changes in the liver and thyroid, and a rat specific change in the kidneys. These findings were considered not to represent an adverse health effect in humans, and hence, the no observed adverse effect level (NOAEL) for male systemic toxicity was established as 100 mg/kg/day.
No test article-related changes were noted for maternal female administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for maternal systemic toxicity, and the subsequent progeny, was established as 100 mg/kg/day.
This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 422 in the rat.
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