Sodium long chain alkyl salicylate

Endpoint summary

Description of key information

ORAL
28 Day systemic NOAEL 100 mg/kg (male and female), rat; OECD 407, EU Method B.7 and US EPA OPPTS 870.3050

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2014 to 07 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality)
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation (mean): Males: 148.75 g; Females: 125.75 g
- Fasting period before study: No, though during motor activity measurements animals had no access to food.
- Housing: Group housing of up to 5 animals per sex in cages (height 18 cm) with sterilised sawdust as bedding material and paper as cage-enrichment. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet except during motor activity measurements
- Water: Free access to tap water except during motor activity measurements
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10 per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
From: No data
To: 07 October 2014

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were stirred during dosing of all animals. Adjustment was made for specific gravity of the vehicle.

DOSE VOLUME: 5 mL/kg bw

VEHICLE
- Specific gravity: 1.036

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CHEMICAL ANALYSIS OF DOSE PREPARATIONS
Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples), samples were stored on dry ice. Samples remained on dry ice until receipt. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Additionally, the long-term stability of the formulation samples prepared in advance of the study and stored at a target temperature =-70 °C was determined. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90 to 110 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.

SAMPLES
In total, 16 samples were included in this study, distributed over 4 formulation groups. The samples of the control group and the 100 mg/kg dose group were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment groups 30 and 300 mg/kg were taken in duplicate from the top, middle and bottom position of the container.
The samples were stored at a target temperature =-70 °C.

ANALYTICAL METHOD
- Analytical conditions
The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
- Sample injections
The analytical run for the determination of the accuracy of preparation and homogeneity of the test material in formulations was acquired in the following order: analytical blank sample, calibration curve, analytical blank sample, analytical blank sample, procedural recovery sample high and low (replicate 1), analytical samples, long term storage stability samples and procedural recovery samples high and low (replicate 2).
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

FORMULAS
Response (Y): Peak area test material [mAU]

Calibration curve: Y = a + bX + cX²
where:
X = nominal concentration [mg/L]
c = curvature
b = slope
a = intercept

Analysed concentration (X): X = [-b + v(b² - 4c(a – y)) / 2c] x ((V x d) / w) [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
[-b + v(b² - 4c(a – y)) / 2c] values are obtained from Analyst® version 1.5.2 (Applied Biosystems, Burlington, Canada).

Recovery: (Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]

Accuracy: (Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

Relative difference (relative diff.): ((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

SPECIFICATIONS
- Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted. Outliers could be discarded as long as minimally 75 % of the responses of the calibration were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 90 to 110 %.
The response in all analytical blank samples at retention time of the test material should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).
- Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 to 110 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.

RESULTS
- Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Regression analysis was performed using the least squares quadratic regression with a (1 / concentration²) weighting factor. The coefficient of correlation (r) was always higher than or equal to 0.9998.
No response at the retention time of the test material in the analytical blank samples was detected.
- Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90 to 110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
- Test samples
In the Group 1 formulations, no test substance was detected. The concentrations analysed in the formulations of Group 2, 3 and Group 4 (dose levels of 30, 100 and 300 mg/kg bw/day respectively) was in agreement with the target concentrations (i.e. mean recoveries between 90 % and 110 %). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10 %).
- Stability at storage conditions
The mean accuracies were 100.2 and 101.3 % for the high (200 mg/g) and the low (1.00 mg/g) concentration levels, respectively, which are within the set criteria (mean accuracy is in the range 90 to 110 %). It was therefore concluded that the test material was stable in propylene glycol over a storage period of at least 25 days at a temperature of =-70 °C.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 30, 100 and 300 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected on the basis of a 14 day range-finding study in which the rats were dosed at 500 and 1000 mg/kg bw.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality and viability were made at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from the start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finding study). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected on the day of necropsy at the end of the treatment phase. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: All animals
- Haematology parameters examined: White blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils and basophils), red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelets.
- Clotting parameters examined: Prothrombin time (PT) and activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected on the day of necropsy at the end of the treatment phase. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: All animals
- Parameters examined: Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium and inorganic phosphate (Inorg. Phos).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 4 of treatment, the following tests were performed on all animals after dosing at no specific time point (but within a similar time period after dosing for all animals).
- Dose groups that were examined: All
- Battery of functions tested: hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R), fore- and hind-limb grip strength (recorded as the mean of three measurements, Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands) and locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerised monitoring system, Kinder Scientific LLC, Poway, USA).

Sacrifice and pathology:

SACRIFICE
On the scheduled day of necropsy, animals were deeply anaesthetised using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied.

PATHOLOGY
Descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10 % buffered formalin (neutral phosphate buffered 4 % formaldehyde solution): Adrenal glands, (aorta), brain [cerebellum, mid-brain, cortex], caecum, cervix, (clitoral gland), colon, duodenum, epididymides*, eyes (including optic nerve [if detectable] and harderian gland)*, (female mammary gland area), femur including joint, heart, ileum, jejunum, kidneys, (larynx), (lacrimal gland, exorbital), liver, lung infused with formalin, lymph nodes - mandibular, mesenteric, (nasopharynx), (oesophagus) , ovaries, (pancreas), peyer's patches [jejunum, ileum] if detectable, (pituitary gland), (preputial gland), prostate gland, rectum, (salivary glands - mandibular, sublingual), sciatic nerve, seminal vesicles including coagulating gland, skeletal muscle, (skin), spinal cord [cervical, midthoracic, lumbar], spleen, sternum with bone marrow, stomach, testes*, thymus, thyroid including parathyroid [if detectable], (tongue), trachea, urinary bladder, uterus, vagina and all gross lesions.
Tissues marked with * were fixed in modified Davidson's solution; tissues were transferred to formalin after fixation for at least 24 hours.
Tissues/organs mentioned in parentheses were not examined by the pathologist.

The following organ weights and terminal body weight were recorded from the animals on the scheduled day of necropsy: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate*, seminal vesicles including coagulating glands* and thyroid including parathyroid.
Organs marked with * were weighed when fixed for at least 24 hours.

HISTOTECHNOLOGY
All organ and tissue samples were processed, embedded in paraffin wax, cut at a thickness of 2 to 4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY
The following slides were examined:
- All tissues collected at the scheduled sacrifice from all control and high dose animals;
- Stomach and liver (males and females) and kidneys (males) of low and mid-dose animals based on (possible) treatment-related changes in these organs in the high dose group; and
- All gross lesions.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data.
Test statistics were calculated on the basis of exact values for means and pooled variances.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Mortality:

mortality observed, treatment-related

Description (incidence):

see below

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No clinical signs of toxicity or abnormalities during weekly arena observations were noted during the observation period.
Slight lethargy, hunched posture and/or piloerection were incidentally observed in males and females at 300 mg/kg/day. Rales were noted in one male at 30 mg/kg/day and one female at 100 mg/kg/day. These clinical signs occurred at a low incidence and/or in single animals only and were therefore considered not toxicologically relevant.
Salivation seen after dosing among animals at 100 and 300 mg/kg/day during the treatment period and seen in females at 30 mg/kg/day for only 2 days was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test material and is considered not toxicologically relevant.

FUNCTIONAL OBSERVATIONS
No toxicologically significant effects on hearing ability, pupillary reflex and static righting reflex were observed. Grip strength was similar between control and high dose animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

BODY WEIGHTS
Slightly lower body weight and body weight gain were noted in males at 300 mg/kg/day.
The slightly lower body weight gain noted in males at 100 mg/kg/day on Days 15 and 22 recovered at the end of the study period and was therefore considered not toxicologically relevant.
The body weight of females at 300 mg/kg/day on Day 29 was only slightly higher compared to control, and considered not toxicologically relevant. No changes in body weight were noted in males and females at 30 mg/kg/day and females at 100 mg/kg/day.

FOOD CONSUMPTION
No toxicologically significant changes in food consumption before or after correction for body weight were noted.

HAEMATOLOGY
Higher white blood cell count was noted in males at 300 mg/kg/day and to a lesser extent also in females at 300 mg/kg/day.
Minor statistically significant changes in mean corpuscular volume (MCV) and platelets count were noted in females at 300 mg/kg/day. These changes were only slight and in absence of changes in related haematology parameters or corroborative microscopic findings, these changes were considered not toxicologically relevant.
Minor statistically significant differences arising between controls and animals receiving 100 mg/kg/day were considered not to represent a change of biological significance.

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters were considered to be related to treatment:
- Higher alanine aminotransferase (ALAT) activity in males and females at 300 mg/kg/day
- Higher alkaline phosphatase (ALP) activity in males and females at 300 mg/kg/day
- Lower total protein level in males at 300 mg/kg/day
- Higher glucose level in males at 300 mg/kg/day
- Lower calcium level in males at 300 mg/kg/day
- Higher cholesterol level in females at 100 and 300 mg/kg/day
Slightly higher aspartate aminotransferase (ASAT) activity was noted in males at 30 and 300 mg/kg/day. These changes occurred in absence of a clear dose-response and were slight in nature.
Therefore these changes were considered not toxicologically relevant.
Any additional statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as these were very slight and occurred in the absence of a dose-related trend.

MACROSCOPIC EXAMINATION
Test item-related irregular surface was observed in the forestomach of one male in the 100 mg/kg/day (1/5) and among males and females in the 300 mg/kg/day group (3/5 males and 3/5 females). The other necropsy findings among control and treated animals were within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend and/or had no treatment-related histopathological correlates. These necropsy findings were therefore considered to be of no toxicological relevance.

ORGAN WEIGHTS
Higher absolute and/or relative liver weights were noted for males and females at 300 mg/kg/day (relative liver weights were increased by 17 % and 15 %, respectively, when compared to mean control levels). Lower absolute and relative thymus weights were noted in males at 300 mg/kg/day (relative 20 %, when compared to control levels). Some organ weight differences were statistically significant when compared to the control group but were considered to be the results of a test item-related effect on final body weight. All other organ weight differences observed, including those that reached statistical significance, were considered incidental and/or related to difference of sexual maturity and unrelated to the treatment.

MICROSCOPIC EXAMINATION
Test item-related microscopic findings after treatment were noted in the forestomach of one 100 mg/kg/day group male and the 300 mg/kg/day group males and females.
Findings in the forestomach were observed in 1/5 males of the 100 mg/kg/day group and 5/5 males and females of the 300 mg/kg/day group and consisted of:
- Granulocytic sub-epithelial inflammation in 1/5 males of the 100 mg/kg/day group (minimal) and in
5/5 males (1 minimal, 3 slight, 1 moderate) and 4/5 females (3 minimal, 1 slight) of the 300 mg/kg/day group
- Hyperkeratosis in 5/5 males (2 slight, 2 moderate, 1 marked) and 5/5 females (2 slight, 3 moderate) of the 300 mg/kg/day group
- Submucosal oedema in 3/5 males (1 minimal, 2 slight) and 3/5 females (all slight) of the 300 mg/kg/day group.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Dose descriptor:

NOAEL

Remarks:

- local

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the morphologic changes in the forestomach, consisting of subepithelial granulocytic inflammation and/or hyperkeratosis and/or submucosal edema, a local No Observed Adverse Effect Level (NOAEL) was established.

Dose descriptor:

NOAEL

Remarks:

- systemic

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the effects on liver, consisting of increases in weight and liver enzyme activities in males and females at 300 mg/kg/day, a systemic No Observed Adverse Effect Level (NOAEL) kg/day was established at 100 mg/kg/day.

Critical effects observed:

not specified

Table 1: Mean Percent Liver and Thymus Weight Differences from Control Groups

		Male			Female
	Dose Level (mg/kg)	30	100	300	30	100	300
Liver	Absolute	-4	-2	6	5	3	23**
	Relative to body weight	1	3	17**	1	3	15**
Thymus	Absolute	-8	-14	-28**	3	2	5
	Relative to body weight	-4	-11	-20*	0	2	-2

*P<0.5;**P=0.01

 

Table 2: Summary Test Material-Related Microscopic Findings – Scheduled Euthanasia Animals (Day 28)

	Male				Female
Dose Level (mg/kg)	0	30	100	300	0	30	100	300
Forestomach*	5	5	5	5	5	5	5	5
Inflammation, granulocytic, sub-epithelial
Minimal	-	-	1	1	-	-	-	3
Slight	-	-	-	3	-	-	-	1
Moderate	-	-	-	1	-	-	-	-
Hyperkeratosis								0
Slight	-	-	-	2	-	-	-	2
Moderate	-	-	-	2	-	-	-	3
Marked	-	-	-	1	-	-	-	-
Oedema, submuscosal
Minimal	-	-	-	1	-	-	-	-
Slight	-	-	-	2	-	-	-	3

* = Number of tissues examined from each group

 

Table 3: Correlations of Selected Observations

Necroscopy	Weight	Clinical Pathology	Histopathology
Forestomach - Irregular surface	-	-	Hyperkeratosis
Liver	Liver ¿	ALAT, ASAT, ALP ¿	Liver -

- = no findings / no correlate

Conclusions:

Based on the morphologic changes in the forestomach, consisting of sub-epithelial granulocytic inflammation and/or hyperkeratosis and/or submucosal oedema, a local No Observed Adverse Effect Level (NOAEL) for the test material of 100 mg/kg/day was established.
Based on the effects on liver, consisting of increases in weight and liver enzyme activities in males and females at 300 mg/kg/day, a systemic No Observed Adverse Effect Level (NOAEL) for EXP1404957 of 100 mg/kg/day was established.

Executive summary:

The repeated dose toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 407, EU Method B.7 and US EPA OPPTS 870.3050 under GLP conditions.

The dose used for the main study was chosen based on the results of a 14-day range finding study; the dose levels for this 28-day oral gavage study were as follows 0, 30, 100 and 300 mg/kg/day.

Study outline

The test material, formulated in propylene glycol, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5males and 5 females.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters were evaluated: clinical signs daily; functional observation tests in Week 4; body weight and food consumption weekly; clinical pathology and macroscopic examination at termination; organ weights and histopathology on a selection of tissues.

Formulation analyses confirmed that formulations of test material in propylene glycol were prepared accurately and homogenously. No mortality occurred in this study and no clinical signs of toxicity or changes in food consumption and functional observations were observed in the treated animals. At 300 mg/kg/day, macroscopic examination of the forestomach showed irregular surface in males and females. The microscopic correlate for this finding was sub-epithelial granulocytic inflammation and/or hyperkeratosis and/or submucosal oedema.

These microscopic findings were considered to reflect damage of the forestomach epithelium by gavage with test material as a bolus and therefore regarded to be adverse in nature at the portal-of-entry. The findings in the forestomach occurred along with slightly lower body weights in males compared to controls.

Organ weight determination revealed test material related higher liver weights in males and females at 300 mg/kg/day. Although no macroscopic and microscopic morphologic alterations in the liver were observed and the changes in liver weights were only minor (15 to 17 % increases in relative liver weight), a higher level of liver enzyme activities, such as alanine aminotransferase and alkaline phosphatase was observed in blood of both sexes. These effects on liver also were supported by higher glucose level and lower total protein level in males, and therefore considered adverse in nature at 300 mg/kg/day.

Other treatment-related changes observed in clinical biochemistry and haematology parameters included higher white blood cell count and lower calcium level in males and higher cholesterol level in females. Test material-related lower thymus weight was noted only in males with no corresponding morphological changes or haematology changes observed.

At 100 mg/kg/day, macroscopic examination revealed irregular surface in the forestomach in one male and microscopic examination revealed granulocytic inflammation in the forestomach of another male. As the degree of severity of granulocytic sub-epithelial inflammation in the forestomach for this male was only minimal, and there were no additional changes in the forestomach either for this male or for the other which presented irregular surface of the forestomach at necropsy, this findings was considered to be not adverse. In females at this dose level only a slightly higher cholesterol level was noted, with no corroborative findings in any other parameters. At 30 mg/kg/day, no treatment-related changes were noted.

Based on the morphologic changes in the forestomach, consisting of sub-epithelial granulocytic inflammation and/or hyperkeratosis and/or submucosal oedema, a local No Observed Adverse Effect Level (NOAEL) of 100 mg/kg/day was established.

Based on the effects on liver, consisting of increases in weight and liver enzyme activities in males and females at 300 mg/kg/day, a systemic No Observed Adverse Effect Level (NOAEL) of 100 mg/kg/day was established.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was conducted in accordance with standardised guidelines under GLP conditions. It was awarded a reliability core of 1 in accordance with the criteria for assessing data quality as set forth by Klimisch et al. (1997). The quality of the database is therefore considered to be good.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subacute Toxicity

The repeated dose toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 407, EU Method B.7 and US EPA OPPTS 870.3050 under GLP conditions.

The dose used for the main study was chosen based on the results of a 14-day range finding study; the dose levels for this 28-day oral gavage study were as follows 0, 30, 100 and 300 mg/kg/day.

Study outline

The test material, formulated in propylene glycol, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5males and 5 females.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters were evaluated: clinical signs daily; functional observation tests in Week 4; body weight and food consumption weekly; clinical pathology and macroscopic examination at termination; organ weights and histopathology on a selection of tissues.

Formulation analyses confirmed that formulations of test material in propylene glycol were prepared accurately and homogenously. No mortality occurred in this study and no clinical signs of toxicity or changes in food consumption and functional observations were observed in the treated animals. At 300 mg/kg/day, macroscopic examination of the forestomach showed irregular surface in males and females. The microscopic correlate for this finding was sub-epithelial granulocytic inflammation and/or hyperkeratosis and/or submucosal oedema.

These microscopic findings were considered to reflect damage of the forestomach epithelium by gavage with test material as a bolus and therefore regarded to be adverse in nature at the portal-of-entry. The findings in the forestomach occurred along with slightly lower body weights in males compared to controls.

Organ weight determination revealed test material related higher liver weights in males and females at 300 mg/kg/day. Although no macroscopic and microscopic morphologic alterations in the liver were observed and the changes in liver weights were only minor (15 to 17 % increases in relative liver weight), a higher level of liver enzyme activities, such as alanine aminotransferase and alkaline phosphatase was observed in blood of both sexes. These effects on liver also were supported by higher glucose level and lower total protein level in males, and therefore considered adverse in nature at 300 mg/kg/day.

Other treatment-related changes observed in clinical biochemistry and haematology parameters included higher white blood cell count and lower calcium level in males and higher cholesterol level in females. Test material-related lower thymus weight was noted only in males with no corresponding morphological changes or haematology changes observed.

At 100 mg/kg/day, macroscopic examination revealed irregular surface in the forestomach in one male and microscopic examination revealed granulocytic inflammation in the forestomach of another male. As the degree of severity of granulocytic sub-epithelial inflammation in the forestomach for this male was only minimal, and there were no additional changes in the forestomach either for this male or for the other which presented irregular surface of the forestomach at necropsy, this findings was considered to be not adverse. In females at this dose level only a slightly higher cholesterol level was noted, with no corroborative findings in any other parameters. At 30 mg/kg/day, no treatment-related changes were noted.

Based on the morphologic changes in the forestomach, consisting of sub-epithelial granulocytic inflammation and/or hyperkeratosis and/or submucosal oedema, a local No Observed Adverse Effect Level (NOAEL) of 100 mg/kg/day was established.

Based on the effects on liver, consisting of increases in weight and liver enzyme activities in males and females at 300 mg/kg/day, a systemic No Observed Adverse Effect Level (NOAEL) of 100 mg/kg/day was established.

Subchronic and Chronic Toxicity

In accordance with Section 8.6.2. of Column 2 of Annex IX of the REACH Regulation, the study does not need to be conducted if a reliable short-term toxicity study (28 days) is available showing severe toxicity effects according to the criteria for classifying the substance as R48, for which the observed NOAEL-28 days, with the application of an appropriate uncertainty factor, allows the extrapolation towards the NOAEL-90 days for the same route of exposure.

 

In such a study, conducted under GLP conditions to EU method B.7/OECD Guideline 407, the NOAEL of the substance was determined to be 100 mg/kg/day and the substance has been classified by the registrant as STOT RE Category 2 and is considered to show sufficient toxicity effects so as to meet the criteria for classifying the substance as R48. This study is considered to allow extrapolation towards the NOAEL-90 for the same route of exposure. As such the registrant proposes to waive the 90-Day Repeat Dose Toxicity study.

 

Furthermore, in accordance with Section 3 of Annex XI, the 90-Day Sub-Chronic Repeat Dose study may be omitted based on exposure scenarios developed in the chemical safety report, given that the comparison of the derived DNEL with the results of the exposure assessment shows that exposures are always well below the derived DNEL. The registrant considers that the grounds for waiving this study are strengthened due to the low potential for exposure to the substance throughout its lifecycle as demonstrated in the chemical safety report and that any potential extra information that could be gained from the performance of this study is outweighed by the opportunity to prevent unnecessary vertebrate animal testing.

Concerning chronic toxicity testing, the substance is not considered to fulfil any of the criteria to propose further testing under Section 8.6.3. of Column 2 of Annex X.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study available

Justification for classification or non-classification

Based on effects seen on the liver at a dose level of 300 mg/kg bw/day, in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance requires classification with respect to specific target organ toxicity after repeated exposure as Category 2, H373: May cause damage to organs (liver).