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EC number: 203-744-6 | CAS number: 110-18-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to reproduction
Administrative data
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The experimental start date was 19 Sep 2017 and the experimental completion date was 07 Aug 2018.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The experimental start date was 19 Sep 2017 and the experimental completion date was 07 Aug 2018.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline 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)
- Version / remarks:
- July 2016
- Deviations:
- yes
- Remarks:
- None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine)
Appearance: Colourless to yellow liquid
Batch: 17F-1068310
Test item storage: At room temperature
Stable under storage conditions until: 30 June 2018 (expiry date) - Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Standard species and strain
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-12 weeks (M) , 12-14 weeks (F)
- Weight at study initiation: 250-350 g (M) , 200-250 g (F)
- Housing: group housed by sex (pre-mating period), cohoused (1:1) during mating, females individually housed during gestation and lactation (with litters)
- Diet: ad libitum pelleted rodent diet
- Water: ad libitum tap water
- Acclimation period: at least 5 days
DETAILS OF FOOD AND WATER QUALITY:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 15/11/2017 To: 19/01/2018 - Route of administration:
- oral: gavage
- Details on route of administration:
- The test item and vehicle were administered by daily gavage for a minimum of 28 days. Males were treated for a minimum of 29 days, up to and including the day before scheduled necropsy. This includes a minimum of two weeks prior to mating and during the mating period. Females were treated for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females were not be dosed during littering. Animals were dosed at approximately at the same time each day The dose volume for each animal was based on the most recent body weight measurement. The dosing formulations were stirred continuously during dose administration.
- Vehicle:
- water
- Details on oral exposure:
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a solution, formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Duplicate middle samples were taken for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples were taken for Groups 2 and 4 (concentration and homogeneity analysis). Stability analyses were performed in conjunction with the method development and validation study.
- Duration of treatment / exposure:
- At least 29 days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Group 1: vehicle control (water)
- Dose / conc.:
- 30 mg/kg bw/day (actual dose received)
- Remarks:
- Group 2
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Group 3
- Dose / conc.:
- 275 mg/kg bw/day (actual dose received)
- Remarks:
- Group 4
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: dose levels were sleceted based on the results of a range-finding study.
- Positive control:
- Not required for this study type
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily (clinical observations); weekly (arena observations)
BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
FOOD CONSUMPTION:
- Time schedule: weekly, except for males and females which are housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
HAEMATOLOGY: Yes
Terminal blood samples were taken at scheduled necropsy of F0 animals (5/sex) for the assessment of haematological parameters (total white blood cells, Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets).
CLINICAL CHEMISTRY: Yes
Terminal blood samples were taken at scheduled necropsy of F0 animals (5/sex) for the assessment of clinical chemistry parameters (Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile Acids, Urea, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)).
Samples were taken at scheduled necropsy of non-selected F0 animals (5/sex) for the assessment of thyroid hormone (T4) and TSH (if required).
Samples were taken from F1 pups at PND4 (2/litter) and PND 13-15 (2/litter) for the assessment of thyroid hormone (T4) and TSH (if required).
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule - once during the treatment period. The selected 5 males were tested once during Week 4 of treatment and the selected 5 females were tested once during the last week of lactation (i.e. PND 6-13). Tests were performed after clinical observations (including arena observation). The following tests were performed:
• Hearing ability, pupillary reflex and static righting reflex
• Fore- and hind-limb grip strength
• Locomotor activity
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. Where no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea recorded.
ORGAN WEIGHTS: Yes
Weights of the brain, cervix, epididymides, adrenals, coagulation gland, parathyroid, prostate, seminal vesicles, thyroid, heart, kidney, liver, ovaries, spleen, testes and thymus were recorded for F0 animals.
HISTOPATHOLOGY: Yes
Histopatholofy was performed on tissues from F0 rats (5/sex) from the control and high dose groups. For the testes of all selected males of Groups 1 and 4, and all males that fail to sire or died before mating detailed qualitative examination were be made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted. - Other examinations:
- OESTRUS CYCLICITY:
Daily vaginal lavage was performed from 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously. - Statistics:
- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and reported at the 1% and 5% levels. Numerical data collected on scheduled occasions were analysed according to sex and occasion. Mean and standard deviation were reported whenever possible. Group means were calculated for continuous data and medians calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations were rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Parametric: datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett's test (many-to-one-t-test).
Non-Parametric: datasets with at least 3 groups were compared using Steel's test (many-to-one rank test). The motor activity data set (at least 3 groups) were compared using an overall Kruskal-Wallis test. Whenever, the overall test was significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
Incidence: An overall Fisher’s exact test was used to compare all groups. The pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- In both males and females salivation was seen after dosing of 275 mg/kg bw/day. This sign is not considered to be toxicologically relevant. From Week 5 of treatment, clinical signs (focal erythema of the ears and hypersensitivity to touch) were noted for most females at 275 mg/kg bw/day.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- One female from Group 4 (275 mg/kg bw/day) was euthanised for humane reasons on Day 12 of treatment. Clinical signs observed in this animal were marked bodyweight loss, hunched posture, laboured breathing, rales and piloerection. Macroscopic findings noted in this animal included distended gastrointestinal tract; small spleen and thymus.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- In males and females, body weights and body weight gains were unaffected by treatmnet during the pre-mating and mating periods. At post coitum Day 4 reduced body weights and body weight gain were observed for females at 100 and 275 mg/kg bw/day; at Day 7 post coitum reduced body weight was observed for females at 275 mg/kg bw/day. At lactation, bodyweights were reduced for females at 275 mg/kg bw/day.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Relative food consumption was reduced for males and females at 275 mg/kg bw/day during the pre mating period. During Days 0 -4 post coitum, food consumption was reduced for females at 275 mg/kg bw/day.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In 275 mg/kg bw/day males, white blood cell count was significantly reduced (by 34%) compared to controls. This findings was considered likely to be due to a decrease in lymphocytes (40% compared to controls). Reticulocyte levels were increased (by 28% compared to controls); MCHC was slightly (4%) but significantly reduced compared to controls. For males at 100 mg/kg bw/day, an increase (19%) in activated partial thromboplastin time was observed; however values are within the historical control data and no dose response relationship was observed. This finding is therefore considered to be unrelated to treatment.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In males at 100 mg/kg bw/day, increased AST (+26% compared to controls) and glucose (+31%) were observed. Bile acids were increased at 100 mg/kg bw/day (+55%) and at 275 mg/kg bw/day (+85%); values do not attain statistical significance. An increase in alkaline phosphatase activity was noted at 100 mg/kg bw/day (+38%) and 275 mg/kg bw/day (+12%). There were no statistically significant changes in females.
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased relative liver weight was seen in males at 100 mg/kg bw/day (+12%) and at 275 mg/kg bw/day (+14%). In females, relative kidney weights were significantly increased at 275 mg/kg bw/day (+17%).
- Gross pathological findings:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- clinical signs
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 275 mg/kg bw/day (nominal)
- System:
- haematopoietic
- Organ:
- blood
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 275 mg/kg bw/day (nominal)
- System:
- nervous system
- Organ:
- brain
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- no
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 275 mg/kg bw/day (nominal)
- System:
- eye
- Organ:
- iris
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- no
- Conclusions:
- Based on the findings of the study, and taking into account effects observed in parental rats, the NOAEL for the systemic toxicity of the substance following 28 days of repeated oral exposure was considered to be 100 mg/kg bw, day, based on histological changes in the brain and eyes (females); focal erythema in both ears, and hypersensitivity to touch (females).
- Executive summary:
The repeated-dose toxicity of ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine) has been investigated in a combined repeated dose/reproductive/developmental toxicity screening test, conducted according to OECD 422 Test Guideline, reliability 1 and in compliance with GLP (Meijer M, 2018). In the study, Wistar Han rats (10 males and 10 females per dose group) were treated with the test substance once daily by oral gavage (seven days per week for a minimum of 28 days) at dose levels of 0, 30, 100 and 275 mg/kg bw/day. Males were treated for 2 weeks prior to mating, during mating, and up to termination (e.g. for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 39-54 days.
No mortality occurred during the study that was considered to be related to treatment with the test item.
One female treated at 275 mg/kg bw/day was sacrificed in extremis on Day 12 of the study due to a dosing error. Adverse parental findings were observed in males and in females dosed at 275 mg/kg bw/day.Females dosed at 275 mg/kg presented with focal erythema of both ears and hypersensitivity to touch from the end of pregnancy onwards. These clinical signs were considered to be adverse considering their high frequency of occurrence and the number of animals affected.
Rales and piloerection observed in several males and females dosed at 275 mg/kg bw/day were not considered adverse, given their low frequency of occurrence.
In the 275 mg/kg bw/day dose group, levels of WBC and lymphocytes were reduced by approximately 34 and 40%, respectively in males and by approximately 41 and 63%, respectively in females. Given the high magnitude of these effects, these changes in leucocyte number were considered to be adverse.
In rats treated at 275 mg/kg bw/day, potentially adverse histopathological changes in the brain and eye consisted of slight hypertrophy of the choroid plexus epithelium and up to moderate vacuolation of the iris. Taking into account that these morphological alterations are rare lesions, the effects on normal functioning of these organs was not readily discernible and it was difficult to conclude on whether these were truly adverse effects or not.
Non-adverse test-item related morphologic alterations were present in the lungs of females (alveolar macrophage aggregations) and in the liver of males (hepatocellular hypertrophy) dosed at 275 mg/kg bw/day. The minimal to slight alveolar macrophage aggregations were not considered to be adverse since there were no other indicators of cellular degeneration or toxicity in the lung. Similarly, the hepatocellular hypertrophy observed in males was minor and in the absence of any other indicator of cellular degeneration, was not considered to be adverse.
A test item-related, but non-adverse, increase in liver weight (relative to body weight) was observed in males treated at 100 and 275 mg/kg (12 and 14%, respectively). No treatment-related changes were noted in any of the remaining repeated-dose parameters investigated in this study (i.e. functional observations, body weight, food consumption, coagulation and clinical biochemistry parameters (including male T4 thyroid hormone levels) and macroscopic examination).
Based on the findings of the study, and taking into account effects observed in parental rats, the NOAEL for the systemic toxicity of the substance following 28 days of repeated oral exposure was considered to be 100 mg/kg bw, day, based on histological changes in the brain and eyes (females); focal erythema in both ears, and hypersensitivity to touch (females).
Table 1. Selected findings in males
Parameter – end of treatment |
Group 1 Control |
Group 2 30 mg/kg bw/d |
Group 3 100 mg/kg bw/d |
Group 4 275 mg/kg bw/d |
|
Haematology (n = 5) |
|||||
WBC 10E9/L |
Mean STDEV |
7.2 2.1 |
6.3 1.0 |
6.8 1.6 |
5.0* 0.9 |
Lymphocytes 10E9/L |
Mean STDEV |
6.7 2.0 |
5.2 0.7 |
5.5 1.4 |
4.0 0.9 |
Reticulocytes 10E9/L |
Mean STDEV |
187.0 26.5 |
206.1 0.28 |
215.4 17.7 |
239.9+ 23.4 |
MCHC mmol/L |
Mean STDEV |
21.78 0.62 |
21.20 0.20 |
21.25 0.43 |
20.95* 0.20 |
Histopathology (n = 5) |
|||||
Liver – hepatocellular hypertrophy |
Minimal |
- |
- |
- |
3 |
+/++ Steel-test significant at 5% (+) or 1% (++) level
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 2. Selected findings in females
Parameter – end of treatment |
Group 1 Control |
Group 2 30 mg/kg bw/d |
Group 3 100 mg/kg bw/d |
Group 4 275 mg/kg bw/d |
|
Haematology (n = 5) |
|||||
WBC 10E9/L |
Mean STDEV |
5.6 1.4 |
5.3 0.6 |
4.9 0.8 |
3.3 2.9 |
Lymphocytes 10E9/L |
Mean STDEV |
4.0 1.1 |
3.7 0.6 |
3.3 0.8 |
1.5+ 1.1 |
Reticulocytes 10E9/L |
Mean STDEV |
216.1 28.8 |
239.3 46.1 |
240.7 17.8 |
220.8 23.4 |
MCHC mmol/L |
Mean STDEV |
20.58 0.55 |
20.87 0.49 |
20.51 0.21 |
20.66 0.26 |
Histopathology (n = 5) |
|||||
Brain – hypertrophy choroid plexus
|
Minimal |
- |
- |
- |
3 |
Slight |
- |
- |
- |
2 |
|
Eyes – vacuolation iris |
Minimal |
- |
- |
- |
1 |
Slight |
- |
- |
- |
2 |
|
Moderate |
- |
- |
- |
1 |
|
Lung – alveolar macrophage aggregation |
Minimal |
- |
- |
1 |
2 |
Slight |
- |
- |
- |
2 |
+/++ Steel-test significant at 5% (+) or 1% (++) level
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 3. Body weight data for males
Body weights (g) – males |
Group 1 Control |
Group 2 30 mg/kg bw/d |
Group 3 100 mg/kg bw/d |
Group 4 275 mg/kg bw/d |
||
Pre mating |
Day 1 Week 1 |
Mean STDEV N |
294 13.4 10 |
292 9.1 10 |
291 7.0 10 |
294 14.7 10 |
Day 8 Week 2 |
Mean STDEV N |
314 18.6 10 |
310 12.9 10 |
309 9.2 10 |
313 21.2 10 |
|
Mating period |
Day 1 Week 1 |
Mean STDEV N |
334 20.4 10 |
329 19.9 10 |
331 9.8 10 |
332 27.5 10 |
Day 8 Week 2 |
Mean STDEV N |
340 21.0 9 |
329 19.9 9 |
331 9.8 10 |
332 27.5 10 |
|
Day 15 Week 3 |
Mean STDEV N |
355 25.8 10 |
344 20.6 10 |
345 11.5 10 |
350 29.0 10 |
Table 4. Body weight data for females
Body weights (g) – females |
Group 1 Control |
Group 2 30 mg/kg bw/d |
Group 3 100 mg/kg bw/d |
Group 4 275 mg/kg bw/d |
||
Pre mating |
Day 1 Week 1 |
Mean STDEV N |
225 6.9 10 |
223 9.8 10 |
223 9.2 10 |
224 11.1 10 |
Day 8 Week 2 |
Mean STDEV N |
231 10.7 10 |
225 8.9 10 |
224 10.7 10 |
224 8.4 10 |
|
Mating period |
Day 1 Week 1 |
Mean STDEV N |
235 10.3 10 |
230 9.2 10 |
227 8.2 10 |
229 11.4 9 |
Day 8 Week 2 |
Mean STDEV N |
|
282 -- 1 |
|
|
|
Day 15 Week 3 |
Mean STDEV N |
|
284 -- 1 |
|
|
|
Day 22 Week 4 |
Mean STDEV N |
|
274 -- 1 |
|
|
|
Day 29 Week 5 |
Mean STDEV N |
|
280 -- 1 |
|
|
|
Day 36 Week 6 |
Mean STDEV N |
|
277 -- 1 |
|
|
- 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:
- The experimental start date was 19 Sep 2017 and the experimental completion date was 07 Aug 2018.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline 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 / Developme ntal Toxicity Screening Test)
- Version / remarks:
- July 2016
- Deviations:
- yes
- Remarks:
- None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine)
Appearance: Colourless to yellow liquid
Batch: 17F-1068310
Test item storage: At room temperature
Stable under storage conditions until: 30 June 2018 (expiry date) - Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River
- Females nulliparous and non-pregnant: yes
- Weight at study initiation: 250-350 g (M) , 200-250 g (F)
- Housing: group housed by sex (pre-mating period), cohoused (1:1) during mating, females individu
ally housed during gestation and lactation (with litters)
- Diet: ad libitum pelleted rodent diet
- Water: ad libitum tap water
- Acclimation period: at least 5 days
DETAILS OF FOOD AND WATER QUALITY:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 15/11/2017 To: 19/01/2018 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a solution, formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Duplicate middle samples were taken for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples were taken for Groups 2 and 4 (concentration and homogeneity analysis). Stability analyses were performed in conjunction with the method development and validation study.
- Details on mating procedure:
- After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- Duration of treatment / exposure:
- At least 29 days
- Frequency of treatment:
- Daily
- Duration of test:
- At least 29 days
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Group 1: vehicle control (water)
- Dose / conc.:
- 30 mg/kg bw/day (actual dose received)
- Remarks:
- Group 2
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Group 3
- Dose / conc.:
- 275 mg/kg bw/day (actual dose received)
- Remarks:
- Group 4
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: dose levels were sleceted based on the results of a range-finding study.
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily (clinical observations); weekly (arena observations)
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
- Time schedule: weekly, except for males and females which are housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0,
4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
Terminal blood samples were taken at scheduled necropsy of F0 animals (5/sex) for the assessment of haematological parameters (total white blood cells, Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets).
Terminal blood samples were taken at scheduled necropsy of F0 animals (5/sex) for the assessment of clinical chemistry parameters (Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile Acids, Urea, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)). Samples were taken at scheduled necropsy of non-selected F0 animals (5/sex) for the assessment of thyroid hormone (T4) and TSH (if required). Samples were taken from F1 pups at PND4 (2/litter) and PND 13-15 (2/litter) for the assessment of thyroid hormone (T4) and TSH.
- Time schedule - once during the treatment period. The selected 5 males were tested once during Week 4 of treatment and the selected 5 females were tested once during the last week of lactation
(i.e. PND 6-13). Tests were performed after clinical observations (including arena observation). The following tests were performed:
• Hearing ability, pupillary reflex and static righting reflex
• Fore- and hind-limb grip strength
• Locomotor activity
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females.
Where no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora
lutea recorded.
Weights of the brain, cervix, epididymides, adrenals, coagulation gland, parathyroid, prostate, seminal vesicles, thyroid, heart, kidney, liver, ovaries, spleen, testes and thymus were recorded for
F0 animals.
Histopatholofy was performed on tissues from F0 rats (5/sex) from the control and high dose groups. For the testes of all selected males of Groups 1 and 4, and all males that fail to sire or died before mating detailed qualitative examination were be made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
OESTRUS CYCLICITY:
Daily vaginal lavage was performed from 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
In summary: the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 14-16 pups, and macroscopy). - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
The organs (ovaries, Uterus, testes) were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanized in extremis. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated. - Fetal examinations:
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. The external reproductive genitals to examine signs of altered development observed. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter was preserved in 10% buffered formalin, the pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.
- Statistics:
- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and reported at the 1% and 5% levels. Numerical data collected on scheduled occasions were analysed according to sex and occasion. Mean and standard deviation were reported whenever possible. Group means were calculated for continuous data and medians calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations were rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Parametric: datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett's test (many-to-one-t-test).
Non-Parametric: datasets with at least 3 groups were compared using Steel's test (many-to-one rank test). The motor activity data set (at least 3 groups) were compared using an overall Kruskal-Wallis test. Whenever, the overall test was significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
Incidence: An overall Fisher’s exact test was used to compare all groups. The pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant. - Indices:
- The following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 14-16 pups, and macroscopy).
- Historical control data:
- Laboratory's historial control data was used
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- In both males and females salivation was seen after dosing of 275 mg/kg bw/day. This sign is not considered to be toxicologically relevant. From Week 5 of treatment, clinical signs (focal erythema of the ears and hypersensitivity to touch) were noted for most females at 275 mg/kg bw/day.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female from Group 4 (275 mg/kg bw/day) was euthanised for humane reasons on Day 12 of treatment due to a dosing error. Clinical signs observed in this animal were marked bodyweight loss, hunched posture, labo
ured breathing, rales and piloerection. Macroscopic findings noted in this animal included distended gastrointestinal tract; small spleen and thymus. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- In males and females, body weights and body weight gains were unaffected by treatmnet during the pre-mating and mating periods. At post coitum Day 4 reduced body weights and body weight gain were observed for females at 100 and 275 mg/kg bw/day; at Day 7 post coitum reduced body weight was observed for females at 275 mg/kg bw/day. At lactation, bodyweights were reduced for females at 275 mg/kg bw/day ,but was not conisdered to be treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Relative food consumption was reduced for males and females at 275 mg/kg bw/day during the pre mating period. During Days 0 -4 post coitum, food consumption was reduced for females at 275 mg/kg bw/day.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In 275 mg/kg bw/day males, white blood cell count was significantly reduced (by 34%) compared to controls. This findings was considered likely to be due to a decrease in lymphocytes (40% compared to controls). Reticulocyte levels were increased (by 28% compared to controls); MCHC was slightly (4%) but significantly reduced compared to controls. For males at 100 mg/kg bw/day, an increase (19%) in activated partial thromboplastin time was observed; however values are within the historical control data and no dose response relationship was observed. This finding is therefore considered to be unrelated to treatment.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In males at 100 mg/kg bw/day, increased AST (+26% compared to controls) and glucose (+31%) were observed. Bile acids were increased at 100 mg/kg bw/day (+55%) and at 275 mg/kg bw/day (+85%); values do not attain statistical significance. An increase in alkaline phosphatase activity was noted at 100 mg/kg bw/day (+38%) and 275 mg/kg bw/day (+12%). There were no statistically significant changes in females.
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased relative liver weight was seen in males at 100 mg/kg bw/day (+12%) and at 275 mg/kg bw/ day (+14%). In females, relative kidney weights were significantly increased at 275 mg/kg bw/day (+17%).
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings were noted in the liver of males treated at 275 mg/kg bw/day and in the brain, eyes and lungs of females treated at 275 mg/kg bw/day. Minimal liver hepatocellular hypertrophy was present in males treated at 275 mg/kg bw/day. This finding corresponded with the increased liver weight observed in male rats treated at 275 mg/kg bw/day, but were not considered to be treatment-related in the absence of any other indicator of cellular degeneration. Increased alveolar macrophage aggregations were present in the lungs of female rates treated at 100 and 275 mg/kg bw/day; these effects were not considered to be treatment-related as no other indicators of cellular degeneration were observed. Slight brain hypertrophy of the choroid plexus and moderate vacuolation of the iris of the eyes was present in females treated at 275 mg/kg bw/day. Taking into account that these morphological alterations are rare lesions, the effects on normal functioning of these organs was not readily discernible and it was difficult to conclude on whether these were truly adverse effects or not.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Details on results:
- Mortality
One female from Group 4 (275 mg/kg bw/day) was euthanised for humane reasons on Day 12 of treatment. Clinical signs observed in this animal were marked bodyweight loss, hunched posture, laboured breathing, rales and piloerection. Macroscopic findings noted in this animal included distended gastrointestinal tract; small spleen and thymus.
Clinical signs
In both males and females salivation was seen after dosing of 275 mg/kg bw/day. This sign is not considered to be toxicologically relevant. From Week 5 of treatment, clinical signs (focal erythema of the ears and hypersensitivity to touch) were noted for most females at 275 mg/kg bw/day.
Functional observations
No effects were observed.
Body weights and food consumption
In males and females, body weights and body weight gains were unaffected by treatmnet during the pre-mating and mating periods. At post coitum Day 4 reduced body weights and body weight gain were observed for females at 100 and 275 mg/kg bw/day; at Day 7 post coitum reduced body weight was observed for females at 275 mg/kg bw/day. At lactation, bodyweights were reduced for females at 275 mg/kg bw/day. Relative food consumption was reduced for males and females at 275 mg/kg bw/day during the pre mating period. During Days 0 -4 post coitum, food consumption was reduced for females at 275 mg/kg bw/day.
Haematology
In 275 mg/kg bw/day males, white blood cell count was significantly reduced (by 34%) compared to controls. This findings was considered likely to be due to a decrease in lymphocytes (40% compared to controls). Reticulocyte levels were increased (by 28% compared to controls); MCHC was slightly (4%) but significantly reduced compared to controls. For males at 100 mg/kg bw/day, an increase (19%) in activated partial thromboplastin time was observed; however values are within the historical control data and no dose response relationship was observed. This finding is therefore considered to be unrelated to treatment.
Clinical biochemistry
In males at 100 mg/kg bw/day, increased AST (+26% compared to controls) and glucose (+31%) were observed. Bile acids were increased at 100 mg/kg bw/day (+55%) and at 275 mg/kg bw/d (+85%); values do not attain statistical significance. An increase in alkaline phosphatase activity was noted at 100 mg/kg bw/day (+38%) and 275 mg/kg bw/day (+12%). There were no statistically significant changes in females.
Macroscopic examination
Gross necropsy did not reveal any toxicologically relevant findings.
Organ weights
Increased relative liver weight was seen in males at 100 mg/kg bw/day (+12%) and at 275 mg/kg bw/day (+14%). In females, relative kidney weights were significantly increased at 275 mg/kg bw/day (+17%). - Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92, 90, 75 and 84% for the control, 30, 100 and 275 mg/kg bw/day groups, respectively.
The low post-implantation survival index noted at 100 mg/kg bw/day (outside the range considered normal for rats of this age ) was mainly attributed to female no. 66 that delivered only 5 pups out of 15 implantation sites. The slightly low post-implantation survival index noted at 275 mg/kg bw/day (at the lower limit considered normal for rats of this age) was mainly attributed to female no. 76 that delivered only 8 pups out of 17 implantation sites. The number of implantation sites and living pups were in normal range.
For female no. 57 (30 mg/kg bw/day), the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study. - Total litter losses by resorption:
- no effects observed
- Description (incidence and severity):
- For female no. 57 (30 mg/kg bw/day), the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study.
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- Litter size was not considered affected by treatment.
Live litter sizes were 10.7, 10.8, 8.6 and 9.7 living fetuses/litter for the control, 30, 100 and 275 mg/kg bw/day groups, respectively. - Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- clinical signs
- haematology
- histopathology: non-neoplastic
- Key result
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: Histopathological changes in the brain and eye (females), decrease in WBC and lymphocytes (both sexes), focal erythema in both ears, and hypersensitivity to touch (females).
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- At 100 and 275 mg/kg bw/day, mean body weights of male and female pups were lower than those of control animals on PND 7 and 13:
- On PND 7, a mean body weight lower than controls of 11% (males) and 13% (females) and of 20% (males) and 19% (females) was recorded for pups at 100 (without statistical significance) and 275 mg/kg bw/day, respectively. The mean values at 275 mg/kg were outside the historical control data (males) or at the lower limit (females) of the range considered normal for pups of this age .
- On PND 13, a mean body weight lower than controls of 15% (males) and 16% (females) and of 23% (males) and 22% (females) was recorded for pups at 100 and 275 mg/kg bw/day, respectively. The mean values at 100 mg/kg bw/day were outside (females) the historical control data or at the lower limit (males) of the range considered normal for pups of this age. The mean values for both sexes at 275 mg/kg were outside the range considered normal for pups of this age.
At 30 mg/kg bw/day, body weights of pups were similar to controls. - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 99, 99, 97 and 97% for the control, 30, 100 and 275 mg/kg bw/day groups, respectively.
One pup of the control group (litter no. 43), one pup at 30 mg/kg bw/day (litter no. 60), two pups at 100 mg/kg bw/day (litter no. 66) and three pups at 275 mg/kg (litter nos. 77 and 80) were found dead at the first litter check. No toxicological relevance was attributed this finding since the mortality incidence/live birth index remained within the range considered normal for pups of this age. - Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- Live litter sizes were 10.7, 10.8, 8.6 and 9.7 living fetuses/litter for the control, 30, 100 and 275 mg/kg bw/day groups, respectively. The lower mean number of living pups (not significant) recorded at 100 mg/kg bw/day (8.6 vs 10.7 in the control group; not statistically significant) was related to the lower post-implantation survival index in this group. As a dose-related response was absent and the mean remained within the range considered normal for rats of this strain and age, this was not considered to be test item-related.
- Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92, 90, 75 and 84% for the control, 30, 100 and 275 mg/kg bw/day groups, respectively.
- External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- no effects observed
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- The viability index at 275 mg/kg bw/day was outside the range considered normal for pups of this age and was potentially affected by treatment. Although a clear dose-related response was absent [viability index in control group was also relatively low (92%) when compared to historical control data (98%)], and in the presence of maternal toxicity at the same dose, a treatment- related effect could not be excluded.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 30 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- fetal/pup body weight changes
- Remarks on result:
- other:
- Remarks:
- Developmental NOAEL: 30 mg/kg bw/day (based on the decrease in body weight of pups on PND 7 and 13).
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 100 mg/kg bw/day
- 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:
- no
- Conclusions:
- Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the developmental toxicity was observed at 100 and 275 mg/kg bw/day.
At 100 and 275 mg/kg bw/day, mean body weights of pups (both sexes) were reduced around 11-13% and 19-20%, respectively on PND 7 and around 15-16% and 22-23%, respectively on PND 13. Given that a clear dose and time-related response was established for this finding and since mean values were outside the range considered normal for pups of this age, these changes in pup body weights were considered adverse.
The viability index at 275 mg/kg bw/day was outside the range considered normal for pups of this age and was potentially affected by treatment. Although a clear dose-related response was absent [viability index in control group was also relatively low (92%) when compared to historical control data (98%)], and in the presence of maternal toxicity at the same dose, a treatment- related effect could not be excluded.
No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, serum levels of T4 thyroid hormone T4 in PND 14-16 pups and macroscopic examination).
No developmental toxicity was observed at 30 mg/kg bw/day. Therefore, no-observed-adverse-effect level (NOAEL) of ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine) was established: 30 mg/kg bw/day (based on the decrease in body weight of pups on PND 7 and 13). - Executive summary:
The developmental toxicity of ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine) has been investigated in a combined repeated dose/reproductive/developmental toxicity screening test, conducted according to OECD 422 Test Guideline, reliability 1 and in compliance with GLP (Meijer M, 2018). In the study, Wistar Han rats (10 males and 10 females per dose group) were treated with the test substance once daily by oral gavage (seven days per week for a minimum of 28 days) at dose levels of 0, 30, 100 and 275 mg/kg bw/day. Males were treated for 2 weeks prior to mating, during mating, and up to termination (e.g. for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 39-54 days.
Regarding general toxicity, in parental rats treated at 275 mg/kg bw/day, potentially adverse histopathological changes in the brain and eye consisted of slight hypertrophy of the choroid plexus epithelium and up to moderate vacuolation of the iris. Taking into account that these morphological alterations are rare lesions, the effects on normal functioning of these organs was not readily discernible and it was difficult to conclude on whether these were truly adverse effects or not. Females dosed at 275 mg/kg presented with focal erythema of both ears and hypersensitivity to touch from the end of pregnancy onwards. These clinical signs were considered to be adverse considering their high frequency of occurrence and the number of animals affected. Based on these findings, the NOAEL for general (systemic) toxicity was determined to be 100 mg/kg bw/day.
Regarding reproductive toxicity, there were no treatment-related changes noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs) observed up to the highest dose level tested (275 mg/kg bw/day).
Based on the findings of this study, the NOAEL for reproductive toxicity was considered to be 275 mg/kg bw/day (i.e. the highest dose tested).
Regarding developmental toxicity: Treatment related effects were observed in the off-spring of rats treated with the substance at 100 and 275 mg/kg bw/day. The mean body weights of pups (both sexes) were reduced around 11-13% and 19-20%, respectively on PND 7 and around 15-16% and 22-23%, respectively on PND 13. Given that a clear dose and time-related response was established for this finding and since mean values were outside the range considered normal for pups of this age, these changes in pup body weights were considered adverse. The viability index at 275 mg/ kg bw/day was outside the range considered normal for pups of this age and was potentially affected by treatment. Although a clear dose-related response was absent [viability index in control group was also relatively low (92%) when compared to historical control data (98%)], and the effects were observed in the presence of maternal toxicity at the same dose, a treatment- related effect could not be excluded.
No treatment related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, serum levels of T4 thyroid hormone T4 in PND 14-16 pups and macroscopic examination). No developmental toxicity was observed at 30 mg/kg bw/day.
Based on the findings of this study, a NOAEL of 30 mg/kg bw/day was established for ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine) for developmental toxicity, based on the decrease in body weight of pups on PND 7 and 13.
Table 1. Developmental data summary
|
Group 1 Control |
Group 2 30 mg/kg bw/d |
Group 3 100 mg/kg bw/d |
Group 4 275 mg/kg bw/d |
Total number of offspring born |
97 |
87 |
79 |
90 |
Total number of uterine implantation sites |
106 |
97 |
105 |
107 |
Number of live offspring on Day 1 after littering |
96 |
86 |
77 |
87 |
Number of live offspring on Day 4 (before culling) |
89 |
86 |
73 |
76 |
Number of live offspring on Day 4 (after culling) |
65 |
61 |
58 |
66 |
Number of live offspring on Day 13 after littering |
65 |
61 |
58 |
66 |
Post-implantation survival index (%) (Total number of offspring born/Total number of uterine implantation sites) * 100 |
92 |
90 |
75 |
84 |
Live birth index (%) (Number of live offspring on Day 1 after littering/Total number of offspring born) * 100 |
99 |
99 |
97 |
97 |
Viability index (%) (Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100 |
93 |
100 |
95 |
87 |
Lactation index (%) (Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100 |
100 |
100 |
100 |
100 |
Table 2. Pup bodyweight (F0 generation) - lactation
Day |
Sex |
|
Group 1 Control |
Group 2 30 mg/kg bw/d |
Group 3 100 mg/kg bw/d |
Group 4 275 mg/kg bw/d |
1 |
M |
Mean STDEV N |
6.2 0.7 9 |
6.4 0.8 8 |
6.1 0.7 8 |
5.7 0.5 9 |
F |
Mean STDEV N |
5.8 0.6 9 |
6.1 0.8 8 |
5.5 0.6 8 |
5.4 0.4 9 |
|
M+F |
Mean STDEV N |
6.0 0.7 9 |
6.3 0.8 8 |
5.8 0.7 9 |
5.6 0.4 9 |
|
4 |
M |
Mean STDEV N |
9.4 1.4 9 |
9.6 1.3 8 |
8.7 1.1 8 |
8.0 0.8 9 |
F |
Mean STDEV N |
9.0 1.4 9 |
9.3 1.4 8 |
8.1 1.4 7 |
7.8 0.7 9 |
|
M+F |
Mean STDEV N |
9.2 1.4 9 |
9.4 1.3 8 |
8.6 1.2 8 |
8.0 0.6 9 |
|
7 |
M |
Mean STDEV N |
15.6 2.2 9 |
15.3 1.6 8 |
13.9 1.5 7 |
12.5** 1.6 9 |
F |
Mean STDEV N |
15.1 1.8 9 |
15.2 1.4 8 |
13.1 2.1 6 |
12.2** 1.3 9 |
|
M+F |
Mean STDEV N |
15.3 1.9 9 |
15.3 1.5 8 |
13.6 1.6 7 |
12.4** 1.3 9 |
|
13 |
M |
Mean STDEV N |
30.4 3.0 9 |
29.4 1.9 8 |
25.9** 2.1 8 |
23.4** 2.5 9 |
F |
Mean STDEV N |
29.5 3.3 9 |
28.9 1.9 8 |
24.9** 1.8 7 |
23.1** 2.3 9 |
|
M+F |
Mean STDEV N |
29.9 3.1 9 |
29.2 1.9 8 |
25.5** 1.8 8 |
23.3** 2.3 9 |
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
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)
- Version / remarks:
- July 2016
- Deviations:
- yes
- Remarks:
- None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- N,N,N',N'-tetramethylethylenediamine
- EC Number:
- 203-744-6
- EC Name:
- N,N,N',N'-tetramethylethylenediamine
- Cas Number:
- 110-18-9
- Molecular formula:
- C6H16N2
- IUPAC Name:
- [2-(dimethylamino)ethyl]dimethylamine
- Test material form:
- liquid
- Details on test material:
- clear, colourless to pale yellow
Constituent 1
- Specific details on test material used for the study:
- ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine)
Appearance: Colourless to yellow liquid
Batch: 17F-1068310
Test item storage: At room temperature
Stable under storage conditions until: 30 June 2018 (expiry date)
Test animals
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Standard species and strain
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-12 weeks (M) , 12-14 weeks (F)
- Weight at study initiation: 250-350 g (M) , 200-250 g (F)
- Housing: group housed by sex (pre-mating period), cohoused (1:1) during mating, females individu
ally housed during gestation and lactation (with litters)
- Diet: ad libitum pelleted rodent diet
- Water: ad libitum tap water
- Acclimation period: at least 5 days
DETAILS OF FOOD AND WATER QUALITY:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 15/11/2017 To: 19/01/2018
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- The test item and vehicle were administered by daily gavage for a minimum of 28 days. Males were treated for a minimum of 29 days, up to and including the day before scheduled necropsy. This includes a minimum of two weeks prior to mating and during the mating period. Females were treated for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females were not be dosed during littering. Animals were dosed at approximately at the same time each day The dose volume for each animal
was based on the most recent body weight measurement. The dosing formulations were stirred continuously during dose administration. - Details on mating procedure:
- Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
A maximum of 14 days were allowed for mating, after which females who had not shown evidence of mating were separated from the males. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Duplicate middle samples were taken for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples were taken for Groups 2 and 4 (concentration and homogeneity analysis). Stability analyses were performed in conjunction with the method development and validation study.
- Duration of treatment / exposure:
- At least 28 days
- Frequency of treatment:
- Daily
- Details on study schedule:
- Litters were culled on PND 4. To reduce variability among the litters, eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples will be collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Group 1: vehicle control
- Dose / conc.:
- 30 mg/kg bw/day (actual dose received)
- Remarks:
- Group 2
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Group 3
- Dose / conc.:
- 275 mg/kg bw/day (actual dose received)
- Remarks:
- Group 4
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Dose selection rationale: dose levels were selected based on the results of a range-finding study
- Positive control:
- Not required for this study type
Examinations
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily (clinical observations); weekly (arena observations)
BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
FOOD CONSUMPTION:
- Time schedule: weekly, except for males and females which are housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
HAEMATOLOGY: Yes
Terminal blood samples were taken at scheduled necropsy of F0 animals (5/sex) for the assessment of haematological parameters (total white blood cells, Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Red blood cells, Reticulo cyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets).
CLINICAL CHEMISTRY: Yes
Terminal blood samples were taken at scheduled necropsy of F0 animals (5/sex) for the assessment of clinical chemistry parameters (Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile Acids, Urea, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)). Samples were taken at scheduled necropsy of non-selected F0 animals (5/sex) for the assessment of thyroid hormone (T4) and TSH (if required).
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule - once during the treatment period. The selected 5 males were tested once during Week 4 of treatment and the selected 5 females were tested once during the last week of lactation (i.e. PND 6-13). Tests were performed after clinical observations (including arena observation). The following tests were performed:
• Hearing ability, pupillary reflex and static righting reflex
• Fore- and hind-limb grip strength
• Locomotor activity
IMMUNOLOGY: No - Oestrous cyclicity (parental animals):
- Daily vaginal lavage was performed from 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
- Sperm parameters (parental animals):
- For the testes of all selected males of Groups 1 and 4, and all males that fail to sire or died before mating detailed qualitative examination were be made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Mortality / moribundity (daily)
Clinical observations (daily)
Bodyweights (PND 1, 4, 7, and 13)
Sex (on PND 1 and 4)
Anogenital distance: anogenital distance (AGD) was measured for all live pups. The AGD was normalized to the cube root of body weight.
Nipple retention: on PND 13, all males in each litter were examined for the number of areola/nipples.
Blood samples were taken from pups on PND 4 (2/litter) and PND 13-15 (2/litter) for the assessment of thyroid hormone (and possible TSH) - Postmortem examinations (parental animals):
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. Where no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea recorded.
ORGAN WEIGHTS: Yes
Weights of the brain, cervix, epididymides, adrenals, coagulation gland, parathyroid, prostate, seminal vesicles, thyroid, heart, kidney, liver, ovaries, spleen, testes and thymus were recorded for F0 animals.
HISTOPATHOLOGY: Yes
Histopathology was performed on tissues from F0 rats (5/sex) from the control and high dose groups. For the testes of all selected males of Groups 1 and 4, and all males that fail to sire or died before mating detailed qualitative examination were be made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted. - Postmortem examinations (offspring):
- Non-selected pups were terminated on PND4 and blood samples taken (2/litter). Remaining pups were sacrificed on PND 13+15 and were assessed for sex and gross abnormalities. Blood samples were taken (2/litter)
- Statistics:
- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and reported at the 1% and 5% levels. Numerical data collected on scheduled occasions were analysed according to sex and occasion. Mean and standard deviation were reported whenever possible. Group means were calculated for continuous data and medians calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations were rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Parametric: datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett's test (many-to-one-t-test).
Non-Parametric: datasets with at least 3 groups were compared using Steel's test (many-to-one rank test). The motor activity data set (at least 3 groups) were compared using an overall Kruskal-Wallis test. Whenever, the overall test was significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
Incidence: An overall Fisher’s exact test was used to compare all groups. The pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant. - Reproductive indices:
- Fertility and gestation indices were calculated.
- Offspring viability indices:
- Live birth, viability (PND4) and lactation (PND13) indices were calculated.
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- In both males and females salivation was seen after dosing of 275 mg/kg bw/day. This sign is not considered to be toxicologically relevant. From Week 5 of treatment, clinical signs (focal erythema of the ears and hypersensitivity to touch) were noted for most females at 275 mg/kg bw/day. These clinical signs were considered to be adverse considering their high frequency of occurence and the number of animals affected.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female from Group 4 (275 mg/kg bw/day) was euthanised for humane reasons on Day 12 of treatment due to a dosing error. Clinical signs observed in this animal were marked bodyweight loss, hunched posture, laboured breathing, rales and piloerection. Macroscopic findings noted in this animal included distended gastrointestinal tract; small spleen and thymus.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In males and females, body weights and body weight gains were unaffected by treatment during the pre-mating and mating periods. At post coitum Day 4 reduced body weights and body weight gain were observed for females at 100 and 275 mg/kg bw/day; at Day 7 post coitum reduced body weight was observed for females at 275 mg/kg bw/day. At lactation, bodyweights were reduced for females at 275 mg/kg bw/day, but was not considered to be treatment-related.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Relative food consumption was reduced for males and females at 275 mg/kg bw/day during the pre-mating period. During Days 0 -4 post coitum, food consumption was reduced for females at 275 mg/kg bw/day.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In 275 mg/kg bw/day males, white blood cell count was significantly reduced (by 34%) compared to controls. This findings was considered likely to be due to a decrease in lymphocytes (40% compared to controls). Reticulocyte levels were increased (by 28% compared to controls); MCHC was slightly (4%) but significantly reduced compared to controls. For males at 100 mg/kg bw/day, an increase (19%) in activated partial thromboplastin time was observed; however values are within the historical control data and no dose response relationship was observed. This finding is therefore considered to be unrelated to treatment.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In males at 100 mg/kg bw/day, increased AST (+26% compared to controls) and glucose (+31%) were observed. Bile acids were increased at 100 mg/kg bw/d (+55%) and at 275 mg/kg bw/day (+85%); values do not attain statistical significance. An increase in alkaline phosphatase activity was noted at 100 mg/kg bw/day (+38%) and 275 mg/kg bw/day (+12%). There were no statistically significant changes in females.
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings were noted in the liver of males treated at 275 mg/kg bw/day and in the brain, eyes and lungs of females treated at 275 mg/kg bw/day. Minimal liver hepatocellular hypertrophy was present in males treated at 275 mg/kg bw/day. This finding corresponded with the increased liver weight observed in male rats treated at 275 mg/kg bw/day, but were not considered to be treatment-related in the absence of any other indicator of cellular degeneration. Increased alveolar macrophage aggregations were present in the lungs of female rates treated at 100 and 275 mg/kg bw/day; these effects were not considered to be treatment-related as no other indicators of cellular degeneration were observed. Slight brain hypertrophy of the choroid plexus and moderate vacuolation of the iris of the eyes was present in females treated at 275 mg/kg bw/day. Taking into account that these morphological alterations are rare lesions, the effects on normal functioning of these organs was not readily discernible and it was difficult to conclude on whether these were truly adverse effects or not.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
Reproductive function / performance (P0)
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- The length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of 4 to 5 days.
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- All females were confirmed to have mated, except for one female at 100 mg/kg bw/day. Precoital time was unaffected by treatment.
Details on results (P0)
One female from Group 4 (275 mg/kg bw/day) was euthanised for humane reasons on Day 12 of treatment. Clinical signs observed in this animal were marked bodyweight loss, hunched posture, laboured breathing, rales and piloerection. Macroscopic findings noted in this animal included distended gastrointestinal tract; small spleen and thymus.
Clinical signs
In both males and females salivation was seen after dosing of 275 mg/kg bw/day. This sign is not considered to be toxicologically relevant. From Week 5 of treatment, clinical signs (focal erythema of the ears and hypersensitivity to touch) were noted for most females at 275 mg/kg bw/day.
Functional observations
No effects were observed.
Body weights and food consumption
In males and females, body weights and body weight gains were unaffected by treatmnet during the pre-mating and mating periods. At post coitum Day 4 reduced body weights and body weight gain were observed for females at 100 and 275 mg/kg bw/day; at Day 7 post coitum reduced body weight was observed for females at 275 mg/kg bw/day. At lactation, bodyweights were reduced for females at 275 mg/kg bw/day. Relative food consumption was reduced for males and females at 275 mg/kg bw/day during the pre mating period. During Days 0 -4 post coitum, food consumption was reduced for females at 275 mg/kg bw/day.
Haematology
In 275 mg/kg bw/day males, white blood cell count was significantly reduced (by 34%) compared to controls. This findings was considered likely to be due to a decrease in lymphocytes (40% compared to controls). Reticulocyte levels were increased (by 28% compared to controls); MCHC was slightly (4%) but significantly reduced compared to controls. For males at 100 mg/kg bw/day, an increase (19%) in activated partial thromboplastin time was observed; however values are within the historical control data and no dose response relationship was observed. This finding is therefore considered to be unrelated to treatment.
Clinical biochemistry
In males at 100 mg/kg bw/day, increased AST (+26% compared to controls) and glucose (+31%) were observed. Bile acids were increased at 100 mg/kg bw/day (+55%) and at 275 mg/kg bw/d (+85%); values do not attain statistical significance. An increase in alkaline phosphatase activity was noted at 100 mg/kg bw/day (+38%) and 275 mg/kg bw/day (+12%). There were no statistically significant changes in females.
Macroscopic examination
Gross necropsy did not reveal any toxicologically relevant findings.
Organ weights
Increased relative liver weight was seen in males at 100 mg/kg bw/day (+12%) and at 275 mg/kg bw/day (+14%). In females, relative kidney weights were significantly increased at 275 mg/kg bw/day (+17%).
Effect levels (P0)
open allclose all
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- haematology
- histopathology: non-neoplastic
- Remarks on result:
- other: Parental NOAEL: 100 mg/kg bw/day, based on the histopathological changes in the brain and eye (females), decrease in WBC and lymphocytes (both sexes), focal erythema in both ears, and hypersensitivity to touch (females).
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 275 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- reproductive function (oestrous cycle)
- reproductive function (sperm measures)
- reproductive performance
- Remarks on result:
- other: No reproductive toxicity was observed up to the highest dose level tested: 275 mg/kg bw/day
Target system / organ toxicity (P0)
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- System:
- other: Brain and eye
- Organ:
- brain
- iris
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- no
Results: P1 (second parental generation)
Reproductive function / performance (P1)
- Reproductive performance:
- no effects observed
Results: F1 generation
General toxicity (F1)
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs occurred among pups that were considered to be related to treatment.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was potentially affected by treatment at 275 mg/kg. Viability indices were 93, 100, 95 and 87% for the control, 30, 100 and 275 mg/kg groups, respectively.
7 pups of the control group (litter nos. 41, 43 and 45), 4 pups at 100 mg/kg (litter nos. 65 and 66) and 11 pups at 275 mg/kg (litter nos. 71, 72, 75 and 76) were missing (most likely cannibalised), spontaneously dead or had to be sacrificed in extremis on PND 1-3.
At 275 mg/kg, mortality incidence between PND 1 and 4 and viability index were outside the range considered normal for pups of this age7. Although a clear dose-related response was absent (mortality incidence between PND 1 and 4 in the control group was relatively high: 0.8 vs 0.2 in our historical control data, but was still within normal range), these dead/missing pups were possibly of toxicological relevance/treatment related.
Female no. 66 at 100 mg/kg had total litter loss on PND 1. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Bodyweights of male and female pups were significantly reduced from PND 13 at 100 mg/kg bw/day and from PND 7 at 275 mg/kg bw/day:
At 100 and 275 mg/kg, mean body weights of male and female pups were lower than those of control animals on PND 7 and 13:
• On PND 7, a mean body weight lower than controls of 11% (males) and 13% (females) and of 20% (males) and 19% (females) was recorded for pups at 100 (without statistical significance) and 275 mg/kg, respectively. The mean values at 275 mg/kg were outside the historical control data (males) or at the lower limit (females) of the range considered normal for pups of this age .
• On PND 13, a mean body weight lower than controls of 15% (males) and 16% (females) and of 23% (males) and 22% (females) was recorded for pups at 100 and 275 mg/kg, respectively. The mean values at 100 mg/kg were outside (females) the historical control data or at the lower limit (males) of the range considered normal for pups of this age8. The mean values for both sexes at 275 mg/kg were outside the range considered normal for pups of this age8.
At 30 mg/kg, body weights of pups were similar to controls. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Serum T4 levels in male and female PND 14-16 pups were not affected by treatment.
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- At 100 and 275 mg/kg, mean body weights of male and female pups were lower than those of control animals on PND 7 and 13:
• On PND 7, a mean body weight lower than controls of 11% (males) and 13% (females) and of 20% (males) and 19% (females) was recorded for pups at 100 (without statistical significance) and 275 mg/kg, respectively. The mean values at 275 mg/kg were outside the historical control data (males) or at the lower limit (females) of the range considered normal for pups of this age .
• On PND 13, a mean body weight lower than controls of 15% (males) and 16% (females) and of 23% (males) and 22% (females) was recorded for pups at 100 and 275 mg/kg, respectively. The mean values at 100 mg/kg were outside (females) the historical control data or at the lower limit (males) of the range considered normal for pups of this age8. The mean values for both sexes at 275 mg/kg were outside the range considered normal for pups of this age8.
At 30 mg/kg, body weights of pups were similar to controls. - Gross pathological findings:
- no effects observed
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Gestation index and duration
Gestation index and the duration of gestation were not considered to be affected by treatment. The gestation indices were 89% for the 30 mg/kg group and 100% for the other groups.
Except for one female at 30 mg/kg (no. 54, with a single implantation site only), all pregnant females had live offspring. The failed pregnancy of female no. 54, without related histopathology changes in reproductive organs, was judged to be unrelated to treatment due to its incidental occurrence and lack of a dose-related trend.
One female at 275 mg/kg (no. 72) delivered after 20 days of gestation. Since the rest of females had a normal duration of gestation (21-22 days), this finding was regarded as incidental and, therefore, considered unrelated to treatment.
Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Post-implantation survival index
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92, 90, 75 and 84% for the control, 30, 100 and 275 mg/kg groups, respectively.
The low post-implantation survival index noted at 100 mg/kg (outside the range considered normal for rats of this age ) was mainly attributed to female no. 66 that delivered only 5 pups out of 15 implantation sites. The slightly low post-implantation survival index noted at 275 mg/kg (at the lower limit considered normal for rats of this age6) was mainly attributed to female no. 76 that delivered only 8 pups out of 17 implantation sites. The number of implantation sites and living pups were in normal range.
For female no. 57 (30 mg/kg), the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study.
Litter size
Live litter sizes were 10.7, 10.8, 8.6 and 9.7 living fetuses/litter for the control, 30, 100 and 275 mg/kg groups, respectively.
The lower mean number of living pups (not significant) recorded at 100 mg/kg (8.6 vs 10.7 in the control group; not statistically significant) was related to the lower post-implantation survival index in this group. As a dose-related response was absent and the mean remained within the range considered normal for rats of this strain and age, this was not considered to be test item-related.
Live birth index
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 99, 99, 97 and 97% for the control, 30, 100 and 275 mg/kg groups, respectively.
One pup of the control group (litter no. 43), one pup at 30 mg/kg (litter no. 60), two pups at 100 mg/kg (litter no. 66) and three pups at 275 mg/kg (litter nos. 77 and 80) were found dead at the first litter check. No toxicological relevance was attributed this finding since the mortality incidence/live birth index remained within the range considered normal for pups of this age
Lactation index
The number of live offspring on PND 13 after littering compared to the number of live offspring on PND 4 (after culling) was not considered to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.
Developmental neurotoxicity (F1)
- Behaviour (functional findings):
- not examined
Developmental immunotoxicity (F1)
- Developmental immunotoxicity:
- not examined
Details on results (F1)
No treatment related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, serum levels of T4 thyroid hormone T4 in PND 14-16 pups and macroscopic examination).
Effect levels (F1)
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 30 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Remarks on result:
- other: Developmental toxicity
- Remarks:
- Developmental NOAEL: 30 mg/kg (based on the decrease in body weight of pups on PND 7 and 13).
Target system / organ toxicity (F1)
- Key result
- Critical effects observed:
- no
Overall reproductive toxicity
- Key result
- Reproductive effects observed:
- no
Any other information on results incl. tables
Table 1. Bodyweight data for females during post-coitum and lactation phases
Body weights (g) – females |
Group 1 Control |
Group 2 30 mg/kg bw/d |
Group 3 100 mg/kg bw/d |
Group 4 275 mg/kg bw/d |
||
Post coitum |
Day 0 |
Mean STDEV N |
236 8.5 9 |
228 8.0 9 |
226 9.7 9 |
226 9.9 9 |
Day 4 |
Mean STDEV N |
251 10.0 9 |
241 7.9 9 |
236** 9.7 9 |
233** 11.1 9 |
|
Day 7 |
Mean STDEV N |
257 11.1 9 |
248 9.4 9 |
242* 7.2 9 |
240** 14.3 9 |
|
Day 11 |
Mean STDEV N |
270 11.4 9 |
261 10.1 9 |
256* 7.8 9 |
258 12.9 9 |
|
Day 14 |
Mean STDEV N |
280 11.8 9 |
270 11.2 9 |
266* 9.8 9 |
269 14.3 9 |
|
Day 17 |
Mean STDEV N |
301 18.8 9 |
292 16.0 9 |
286 11.2 9 |
286 19.8 9 |
|
Day 20 |
Mean STDEV N |
338 20.1 9 |
323 24.8 9 |
319 16.0 9 |
319 18.9 9 |
|
Lactation |
Day 0 |
Mean STDEV N |
265 14.3 9 |
257 9.7 8 |
251 9.8 9 |
239** 15.3 9 |
Day 4 |
Mean STDEV N |
272 10.5 9 |
263 13.6 8 |
259 10.4 8 |
244** 14.7 9 |
|
Day 7 |
Mean STDEV N |
276 13.4 9 |
271 10.7 8 |
265 11.0 8 |
258* 15.7 9 |
|
Day 13 |
Mean STDEV N |
286 13.6 9 |
288 11.2 8 |
274 6.1 8 |
277 13.8 9 |
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 2. Reproduction data summary
|
Group 1 Control |
Group 2 30 mg/kg bw/d |
Group 3 100 mg/kg bw/d |
Group 4 275 mg/kg bw/d |
Females paired |
10 |
10 |
10 |
9 |
Females mated |
10 |
9 |
10 |
9 |
Pregnant females |
9 |
9 |
9 |
9 |
Females with implantations only |
0 |
1 |
0 |
0 |
Females with total litter loss on PND 1 |
0 |
0 |
1 |
0 |
Females with living pups on Day 1 |
9 |
8 |
9 |
9 |
Mating index (%) (Females mated / Females paired) * 100 |
100 |
90 |
100 |
100 |
Fertility index (%) (Pregnant females / Females mated) * 100 |
90 |
100 |
90 |
100 |
Gestation index (%) (Females with living pups on Day 1 / Pregnant females) * 100 |
100 |
89 |
100 |
100 |
Applicant's summary and conclusion
- Conclusions:
- In a combined repeated dose/reproductive/developmental toxicity screening test, conducted according to OECD 422 Test Guideline, reliability 1 and in compliance with GLP, the following NOAEL of ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine) were established:
Parental NOAEL: 100 mg/kg bw /day, based on the histopathological changes in the brain and eye (females), decrease in WBC and lymphocytes (both sexes), focal erythema in both ears, and hypersensitivity to touch (females).
Reproduction NOAEL: At least 275 mg/kg bw/day.
Developmental NOAEL: 30 mg/kg bw/day (based on the decrease in body weight of pups on PND 7 and 13). - Executive summary:
The reproductive toxicity of ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine) has been investigated in a combined repeated dose/reproductive/developmental toxicity screening test, conducted according to OECD 422 Test Guideline, reliability 1 and in compliance with GLP (Meijer M, 2018). In the study, Wistar Han rats (10 males and 10 females per dose group) were treated with the test substance once daily by oral gavage (seven days per week for a minimum of 28 days) at dose levels of 0, 30, 100 and 275 mg/kg bw/day. Males were treated for 2 weeks prior to mating, during mating, and up to termination (e.g. for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 39-54 days.
Regarding general toxicity, in parental rats treated at 275 mg/kg bw/day, potentially adverse histopathological changes in the brain and eye consisted of slight hypertrophy of the choroid plexus epithelium and up to moderate vacuolation of the iris. Taking into account that these morphological alterations are rare lesions, the effects on normal functioning of these organs was not readily discernible and it was difficult to conclude on whether these were truly adverse effects or not. Females dosed at 275 mg/kg presented with focal erythema of both ears and hypersensitivity to touch from the end of pregnancy onwards. These clinical signs were considered to be adverse considering their high frequency of occurrence and the number of animals affected. Based on these findings, the NOAEL for general (systemic) toxicity was determined to be 100 mg/kg bw/day.
Regarding reproductive toxicity, there were no treatment-related changes noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs) observed up to the highest dose level tested (275 mg/kg bw/day).
Based on the findings of this study, the NOAEL for reproductive toxicity was considered to be 275 mg/kg bw/day (i.e. the highest dose tested).
Regarding developmental toxicity: Treatment related effects were observed in the off-spring of rats treated with the substance at 100 and 275 mg/kg bw/day. The mean body weights of pups (both sexes) were reduced around 11-13% and 19-20%, respectively on PND 7 and around 15-16% and 22-23%, respectively on PND 13. Given that a clear dose and time-related response was established for this finding and since mean values were outside the range considered normal for pups of this age, these changes in pup body weights were considered adverse. The viability index at 275 mg/ kg bw/day was outside the range considered normal for pups of this age and was potentially affected by treatment. Although a clear dose-related response was absent [viability index in control group was also relatively low (92%) when compared to historical control data (98%)], and the effects were observed in the presence of maternal toxicity at the same dose, a treatment- related effect could not be excluded.
No treatment related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, serum levels of T4 thyroid hormone T4 in PND 14-16 pups and macroscopic examination). No developmental toxicity was observed at 30 mg/kg bw/day.
Based on the findings of this study, a NOAEL of 30 mg/kg bw/day was established for ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine) for developmental toxicity, based on the decrease in body weight of pups on PND 7 and 13.
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