Registration Dossier

Diss Factsheets

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

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).

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

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

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
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

Constituent 1
Chemical structure
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
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)

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%).

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)

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).

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.