N,N,N',N'-tetramethylethylenediamine

Endpoint summary

Description of key information

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.  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).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

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

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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was reliabilty 1, conducted according to the Test Guideline and in compliance with GLP.

System:

other:

Organ:

brain

iris

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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).

Justification for classification or non-classification

Based on the available data, the substance ATMEDAHP (N,N,N’,N’-tetramethylethylenediamine) does not require classification for repeated dose toxicity according to Regulation (EC) 1272/2008.