Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-21 to 2017-03-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
revised 1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P)14-15 wks
- Weight at study initiation: (P) Males: 371-434 g; Females: 231-290 g
- Housing: individually during premating period for both sexes and during postmating for males depending on mating status, IVC cages (type III H, polysulphone cages)
- Diet (e.g. ad libitum): ad libitum; Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): ad libitum, tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals).
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulation was prepared once a week based on results obtained from stability investigations. The test item formulation was stored at approximately -20 °C and was thawed at room temperature before each administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test item’s characteristics and on results obtained from another study
- Concentration in vehicle: 2, 6 and 12 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 57106885543
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: for maximum 14 days/ until mating
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
DETAILS ON PRETREATMENT
- Sample storage conditions before analysis: After receipt at the Analytics Department samples were stored at -15 to -35°C until analysis. After analysis samples are kept stored at -15 to -35°C until at least 3 months after finalization of phase report.

IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE/PRODUCT
- Separation method (e.g. HPLC, GC): HPLC UV
- Conditions (column, mobile phase, etc.): Column: Atlantis T3 3µm, 3 x 50 mm, Waters 186003721, Nr. 48, Solvent A: Water, Solvent B: Acetonitrile, Injection volume: 6 µL, Flow rate: 0.7 mL/min
- Detection method (e.g. ECD, UV, MS, ICP-AES, ICP-MS): UV

Concentration analysis of formulation samples was determined at three concentrations, 2.0 mg/mL, 6.0 mg/mL and 12.0 mg/mL in study weeks 1, 3, 5 and 7. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 103.7%, 99.7% and 99.5% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups in study week 1 and study week 7 as measured concentrations were within acceptance criterion of 15%. In study week 3 however, the single samples no.19 (MD) did not meet the acceptance criteria as recovery was lower than 15% of nominal concentration. In study week 5 the single samples no.22 (LD) did not meet the acceptance criteria as recovery was higher than 15% of nominal concentration. These results were verified by reanalysis.
Homogeneity of formulation samples was determined at two concentrations, 2.0 mg/mL and 12.0 mg/mL, in study weeks 1 and 5 of the study. The mean recoveries observed for the LD dose group was 109.1% and 115.3% of the nominal value and 105.2% and 99.2% of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 3.1% and 2.0% in LD dose group, 0.4% and 1.3% in HD dose group. All samples were homogenous, as COV was below or equal 15%.
Duration of treatment / exposure:
Male: 28 days, i.e. 14 days prior to mating and 14 days during mating
Female: maximum 63 days, i.e. 14 days prior to mating, 14 days during mating and during gestation upto postnatal day 12.
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 16-17 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Results of a dose range finding study
Positive control:
no

Examinations

Parental animals: Observations and examinations:
MORTALITY:Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed once before assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. Females were weighed on gestation day 0, 7, 14 and 20 and within 24 h post partum, i.e., on post partum day 0 and on post partum day 4 and 13 along with their pups.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/week: Yes
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily animals were observed for morbidity and mortality, except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were made once a day

CLINICAL BIOCHEMISTRY: Yes
- Time schedule: From all dams and 2 pups/litter at termination on day 13 and from all adults males at termination, blood samples were collected from the defined site in serum separator tubes.
Oestrous cyclicity (parental animals):
Estrous cycles were monitored before treatment initiation to select for the study females with regular estrous cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
histopathological examination taking into account the tubular stages of the spermatogenic cycle
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

CLINICAL BIOCHEMISTRY
From 2 females pups wherever possible/litter on day 4 after birth; from 2 pups/litter at termination on day 13 blood samples were collected from the defined site in serum separator tubes. Subsequently, the blood samples were assessed for serum levels for thyroid hormones (T4).
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at the end of the mating period, i.e. at day 29 and day 30
- Maternal animals: All surviving animals along with their pups on postnatal day 13

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

- Gross necropsy consisted of external and internal examinations including the thoracic and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.
Statistics:
A statistical assessment of the results of the body weight, food consumption and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).
Reproductive indices:
Copulation Index (%) = (No. of rats copulated / No. of pairs) X 100
Fertility Index (%) = (No. of females pregnant / No. of females copulated) X 100
Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) X 100
Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100
Offspring viability indices:
No. of Male (live & dead)
No. of Female (live & dead)
Sex ratio (m/f, live & dead)
Live pups
Still birth
Runt

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- no toxicologically relevant clinical signs were noted
- In males, predominant clinical signs observed during the treatment period (PMD 11 to MD 8 ) were diarrhoea in Control (C) and Low Dose (LD) group and both diarrhoea and moving the bedding in Mid Dose (MD) and High Dose (HD) group. Isolated incidences of piloerection, reduced spontaneous activity, abnormal breathing and red nasal discharge in very few male animals of LD, MD and HD.
In females, predominant clinical signs observed during the treatment period (PMD 11 to PND 12 ) were diarrhoea in C and LD group and both diarrhoea and moving the bedding in MD and HD group. Isolated incidences of alopecia on various body parts, pale skin, salivation, vocalisation, piloerection, abnormal breathing, reduced spontaneous activity, wasp waist and red nasal discharge in very few female animals of all groups including control group were considered to be incidental. None of the females showed signs of abortion or premature delivery.

Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- no test item-related mortality occurred during the study period.
- During the treatment period of this study, Female no. 44 Control (C) was found dead on premating day (PMD) 13. Female no. 69 (Mid Dose group) was found dead on gestation day (GD) 2.
Male no. 32 High Dose group (HD) was found dead on mating and post mating day (MD) 3. Male no. 35 (HD) was euthanised in moribund condition on PMD 8 due to animal welfare reasons.
Female no. 58 Low Dose group (LD) was euthanised in moribund condition on post-natal day (PND) 0 due to animal welfare reasons. Histopathologically, the cause of death/morbidity in animal no. 32 (HD) and 58 (LD) was considered to be due to gavaging error (tracheal inflammation, thymic inflammation with plant particles). The cause of death/morbidity could not be established for the other animals.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase in group mean food consumption in female Mid Dose group was observed during premating day 7-14 when compared with the controls. Due to lack of dose dependency and consistency, this statistically significant effect on female food consumption was considered to be incidental and not related to the treatment with test item.
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:
no effects observed
Description (incidence and severity):
No test item related effect of toxicological relevance was observed on pup thyroid weight and pup thyroxine hormone (T4) in the treatment groups when compared to the controls.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In males, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group. However, there was a moderately lower absolute and relative mean weight of levator ani and bulbuocavernosus muscle complex in the male treatment groups (lower by approx. 8-15% vs controls) when compared with the controls. As no macroscopic findings were observed and in the absence of statistical significance, these changes were not considered to be of toxicological relevance. In females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance. The macroscopic changes observed were abnormal color red and gas filled stomach, dark red lung and digestive tract- gas filled (male no. 32 of HD), dark red lung (female no. 69 of MD), dark abnormal colored uterus and fluid filled oviduct, stomach (female No. 58 from LD) and red fluid filled thoracic cavity (female 44 of Control and 69 of MD group). These changes were within the range of normal background alterations which may be recorded in animals of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Gross lesions were unremarkable and not related to treatment with the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The test item had no biologically significant effect on the estrous cycle analyzed during the 2-week premating period after the first administration. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed occasionally in few animals of all treatment groups including control. As this effect was also observed in control animals, it was considered as biological variation and not related to the treatment with the test item.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Testis weight, epididymis weight and histopathological examination taking into account the tubular stages of the spermatogenic cycle were determined. There were no remarkable effects.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slightly reduced fertility index (number of females pregnant / number of females copulated X 100) of 80 % in the MD group compared to 90 % in all other groups. In the absence of dose response dependency, the finding was not considered to be of toxicological relevance.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity and fertility
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect observed up to the highest dose tested

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item treatment related effects on litter data including total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
A marginally higher mean mortality of pups between PND 4 and PND 13 was observed in the HD group (1.01%) compared to the control group (0.00%). This outcome did not achieve statistical significance and was attributed to missing one single pup of one
single dam (pup no. 2 of dam no. 78) on PND 8. This effect was considered as incidental and not related to the treatment with the test item. There were also few mortalities/missing pups observed from LD and MD group, and as a result a higher mean mortality of pups between PND 0 and PND 4 was observed in the LD and MD groups (2.81 and 0.89 %, respectively) compared to the control group (0.00%). Due to lack of dose dependency and consistency, this effect in LD and MD group was not considered to be treatment related.
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
not examined
Description (incidence and severity):
No test item related effect of toxicological relevance was observed on anogenital distance and nipple retention in the pups of any of the groups. However, statistically significantly lower male pup weight, cube root of male pup weight and relative anogenital distance of male pups was observed in MD group when compared with the controls. Due to lack of dose dependency and consistency, this effect in MD group was not considered to be treatment related.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related gross external abnormalities of toxicological relevance on PND 0 were observed in the pups of any of the groups.
Few specific findings like wound on left knee on PND 2 (pup no 10 from dam 54) and dark scruff on PND 0 (pup no. 7 from dam 54) were observed in LD group. Female number 58 from LD group was euthnaised for animal welfare reasons on PND 0 after littering and 10 out of 12 pups were still born and other 2 pups were euthanised with dam due to welfare reasons. On PND 0, these pups were observed with predominant external findings like dark head, bloody mouth and few were partly cannibalised.
The external findings slight growth of coat at back (pup 2 and 6 from dam 59 of LD group) crushed pup (pup 13 from dam 64 of MD group) and small- thin pups (pup 10 and 13 from dam 66) at death were considered to be spontaneous and not related to test item treatment.
Histopathological findings:
no effects observed
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no treatment-related effects observed up to the highest dose tested

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:Mortality and clinical signs

 

Observation

Dose Group

Control

LDT

MDT

HDT

[P]Generation - Males

 Mortality

 0/10

0/10 

0/10 

 2/10

 

 

 

found dead: no. 32 (MD 3)

euthanasia: no. 35 (PMD 8)

abnormal breathing (moderate)

-

-

-

2/10

abnormal breathing (severe)

-

-

-

1/10

diarrhoea

10/10

10/10

10/10

9/10

moving the bedding

-

-

10/10

10/10

nasal discharge (red)

-

2/10

3/10

1/10

piloerection (slight)

-

-

-

1/10

spontaneous activity reduced (moderate)

-

-

-

1/10

 

 

 

 

 

[P]Generation - Females

 Mortality

1/10 

1/10 

1/10 

0/10 

 

found dead: no. 44 (PMD 13) 

euthanasia: no. 58 (PND 0) 

found dead: no. 69 (GD 2) 

 

abnormal breathing

1/10

1/10

1/10

4/10

alopecia (back)

-

1/10

-

-

alopecia (both forepaws)

1/10

-

-

2/10

alopecia (shoulders)

-

-

1/10

-

alopecia (throat bilateral)

-

-

3/10

-

alopecia (throat left)

-

-

1/10

-

diarrhoea

10/10

10/10

10/10

10/10

moving the bedding

-

3/10

9/10

10/10

nasal discharge (red)

1/10

-

-

1/10

pale skin

-

1/10

-

-

piloerection (slight)

-

2/10

1/10

2/10

piloerection (moderate)

-

-

1/10

1/10

salivation (slight)

1/10

-

1/10

3/10

salivation (moderate)

1/10

1/10

-

4/10

salivation (severe)

-

-

-

2/10

spontaneous activity reduced (moderate)

-

1/10

-

-

vocalisation

-

-

-

1/10

wasp waist (moderate)

-

1/10

-

-

Animals affected / total number of animals; Given in brackets: study day of death PMD = pre mating day; PND = post-natal day; GD = gestation day, MD- mating and post mating day

a        Data extracted from pages (38) of the study report

Table 3: Pre- and Post-Natal Data - Summary

Group

 

Corpora

Lutea

(CL)

Implantation Sites (IS)

Live

Pups on PND 0

Live

Pups on

PND 4

(before)

Live

Pups on

PND 4

(after)

Live

Pups on

PND 13

Pre

Implantation Loss (%)

Post

Implantation Loss (%)

C

Mean

12.89

12.00

10.89

10.89

8.89

8.89

6.71

9.72

SD

1.17

1.12

1.90

1.90

1.90

1.90

6.75

9.48

N

9

9

9

9

9

9

9

9

LD

Mean

12.25

12.25

9.44

10.13

8.13

8.13

0.00

15.20

SD

2.12

2.12

3.64

2.70

2.70

2.70

0.00

15.48

N

8

8

9

8

8

8

8

8

MD

Mean

13.25

12.75

11.88

11.75

9.88

9.75

3.59

6.74

SD

1.91

1.75

1.81

1.67

1.81

1.67

5.16

7.38

N

8

8

8

8

8

8

8

8

HD

Mean

13.89

13.11

12.00

12.00

10.00

9.89

5.16

8.51

SD

1.45

0.93

1.32

1.32

1.32

1.27

6.40

7.16

N

9

9

9

9

9

9

9

9

 

Table 4:Litter parameters for F1

 

Observation

Dose Group (ppm)

Control

LDT

MDT

HDT

F1Generation

Mean Implantation Sites

12.00 

12.25 

12.75 

13.11 

Number born live

10.89 

9.44 

 11.88

12.00 

Pre Implantation loss [%]

6.71 

0.00 

 3.59

 5.16

# Deaths    Days 0-4 (%)

 0.00

2.81 

0.89 

0.00

# Deaths    Days 4-13 (%)

0.00 

0.00 

0.00 

 1.01 

Mean litter size Day 0

10.89 

 10.38

11.88 

12.00 

Day 4

10.89 

10.13 

11.75 

12.00 

Day 13

8.89 

 8.13

9.75

9.89 

 

 

 

 

aData extracted from pgs (56-58) of the study report.

Table 5: Mean Anogenital Distance and Nipple Retention

Group

 

Male Pups

Male Pup Weight (g)

Cube Root of Pup

Weight

Anogenital

Distance

(mm) of Pups

Relative

Anogenital

Distance of Pups

Pup nipple retention

(N) on PND 12

Female Pups

Female

Pup

Weight (g)

Cube

Root of Pup

Weight

Anogenital

Distance (mm) of Pups

Relative

Anogenital

Distance Pups

C

Mean

3.47

6.61

1.87

1.95

1.04

0.02

8.79

6.23

1.84

0.66

0.34

SD

2.06

0.76

0.08

0.37

0.18

0.14

2.23

0.60

0.06

0.23

0.07

N

51

51

51

51

51

51

47

47

47

49

47

LD

Mean

3.46

6.29

1.84

1.92

1.05

0.00

8.53

5.84

1.80

0.71

0.35

SD

2.02

0.85

0.09

0.27

0.13

0.00

2.73

0.71

0.07

0.41

0.07

N

46

45

45

49

45

44

40

40

40

48

40

MD

Mean

4.54

6.15**

1.83**

2.04

1.12*

0.04

9.53

6.06

1.82

0.65

0.35

SD

2.85

0.76

0.07

0.32

0.16

0.19

2.48

0.86

0.09

0.15

0.08

N

56

57

57

57

57

56

38

38

38

39

38

HD

Mean

3.81

6.31

1.84

1.97

1.07

0.29

9.20

5.99

1.81

0.64

0.35

SD

2.21

0.82

0.08

0.28

0.13

0.83

2.40

0.76

0.08

0.12

0.07

N

52

52

52

53

52

51

56

56

56

56

56

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

Applicant's summary and conclusion

Conclusions:
On the basis of this reproduction/developmental toxicity screening test with 2,4-Bismaleimidotoluene in male and female Wistar rats with dose levels of 10, 30, and 60 mg/kg body weight day the following conclusion can be made: No adverse effects of 2,4-Bismaleimidotoluene were found on male, females and pups at dose levels of 60 mg/kg body weight.
The NOAEL of 2,4-Bismaleimidotoluene in this study for general and reproductive toxicity screening is considered to be 60 mg/kg body weight.

Based on these results, the following NOAELs were derived:
parental NOAEL: 60 mg/kg
fertility NOAEL, females: 60 mg/kg
developmental NOAEL: 60 mg/kg