Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Two generation study; oral (gavage), rat (Wistar rats Crl: (WI) BR; 24/sex/dose), OECD TG 416, GLP; NOAEL (parental) = 6 mg a.i./kg bw/d, based on decreases in body weight, body weight gain and relative food consumption
NOAEL(reproduction) = 6 mg a.i./kg bw/d, based on reduced number of implantation sites and living pups at day 1 of lactation in P0-dams
NOAEL(development) = 20 mg a.i./kg bw/d, no effects

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
2001
Deviations:
yes
Remarks:
the conduct of the study was consistent in all important aspects to the OECD 416 (2001) except that the examination of the oestrus cycle and sperm parameters are missing.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar rats Crl: (WI) BR (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: P-females: 15 weeks, P-males: 7 weeks
- Weight at study initiation: P-females: 219–299g, P-males: 236–292g
- Housing: premating - in groups of max. 4; during mating - 1:1; after mating - individually; offsprong were kept with the dam until weaning
- Diet: standard pelleted diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13-20 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
water
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 3 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after three unsuccessful weeks: no
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared during weeks 1, 8, 13, 22 and 36 were analysed to check homogeneity and accuracy of preparation (all concentrations).
Analysis of the formulations revealed values for accuracy within the range of 92% and 105% of nominal. This was considered to represent an acceptable level for formulations of this type. The low and high dose formulations were considered to be sufficiently homogenous to be used in this type of study. Several measurements were considered to be outliers due to malfunctioning of the TOC apparatus, and excluded from interpretation.
Duration of treatment / exposure:
P-males: 10 weeks prior to mating up to termination.
P-females: 2 weeks prior to mating, during mating, pregnancy and lactation.
F1-males: from weaning onwards for at least 10 weeks prior to mating up to termination.
F1 females: from weaning onwards for at least 10 weeks prior to mating, during mating, pregnancy and lactation.
F2-pups were not treated.
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until at least 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation after weaning.
- Age at mating of the mated animals in the study: at least 10 weeks
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
based on test substance as manufactured (20% aqueous solution)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
based on test substance as manufactured (20% aqueous solution)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
based on test substance as manufactured (20% aqueous solution)
Dose / conc.:
2 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
6 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
No. of animals per sex per dose:
P-generation: 24 animals of each sex per group
F1-generation: 22 to 24 animals of each sex per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on an embryotoxicity and teratogencity study with the registration substance (20% solution) admininstered by oral gavage in female Wistar rats, in which 0, 40, 100, and 250 mg/kg bw/day were dosed. Females of the 250 mg/kg bw/day dose group showed clinical signs, and reduced body weight, body weight gain, food consumption and relative food consumption when compared to the control group. No effects were seen in the 40 and 100 mg/kg bw/day groups.
Positive control:
none
Parental animals: Observations and examinations:
Clinical signs: Yes, daily

Body weight: Yes; weekly
Mated females were weighed on gestation days 0, 7, 14 and 21 and during lactation on the same days as the weighing of the litters (days 1, 4, 7, 14, 21 and 25).

Food consumption: Yes, weekly
Food consumption of mated females was measured on gestation days 0, 7, 14 and 21 and during lactation on the same days as the weighing of the litters (days 1, 4, 7, 14, 21 and 25).

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation performed since no effect on water consumption was suspected.

Oestrous cyclicity (parental animals):
Not determined
Sperm parameters (parental animals):
Not determined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death wasnot determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
F0-males were killed as soon as possible after confirmation of pregnancy of F0-females. F0-females were killed as soon as possible after the lactation period.
On day 4 of lactation or shortly thereafter, F1-offspring and F2-offspring eliminated for standardisation of the litter were killed. F1-offspring not selected for mating were killed as soon as possible after weaning. F1-offspring selected for mating were killed as soon as possible after weaning of the F2-offspring.

MACROSCOPIC EXAMINATION
All parental animals (P and F1) were subjected to macroscopic examination of the cranial, thoracic, abdominal tissues and organs, with special attention to the reproductive organs and abnormalities.
Samples of the following tissues and organs were fixed in 4% formaldehyde: all gross lesions, cervix, coagulation gland, epididymides, ovaries, pituitary gland, prostate, seminal vesicles, stomach, testes, uterus, vagina.
Postmortem examinations (offspring):
F1-offspring not selected for mating and F2-offspring:
Offspring found dead or killed before day 14 of lactation were sexed and externally examined if practically possible. If possible, defects or cause of death were evaluated. The stomach was examined for the presence of milk. No pups were preserved for further examination.
Offspring found dead or killed on or after day 14 of lactation were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs. Descriptions of all macroscopic abnormalities were recorded. If possible, defects or cause of death were evaluated. No pups were preserved for further examination.
Statistics:
Body weight, body weight gain, food consumption, relative food consumption, pup weight: Dunnett-Test (variables could be assumed to follow a normal distribution)
Implantation sites, living pups at 1st litter check: Steel-test (data cannot be assumed follow a normal distribution)
Macroscopic findings, fertility index, conception rate, gestation index, percentage mating, breeding data: Fisher’s Exact Test
Reproductive indices:
Percentage mating
Fertility index
Conception rate
Gestation index
Offspring viability indices:
Percentage live males at first litter check
Percentage live females at first litter check
Percentage of postnatal loss days 0-4 post partum
Percentage of breeding loss day 5 until weaning
Percentage live males at weaning
Percentage live females at weaning
Viability index
Weaning index
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rales were observed at a low incidence and on few occasions among the high dosed animals; the high dosed females showed a slightly increased incidence of hunched posture. No nodules or masses were noted.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality was observed at any dose level.
One female rat from the 10 mg/kg bw/day dose group died spontanously on day 40 due to delivery difficulites. 1 male and 1 female rat from the 100 mg/kg bw/day dose group were found dead. One male rat from the high dose group was killed in extremis due to bad health.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were decreased in:
- Males (100 mg/kg bw/day) from the 5th week of treatment onwards, which showed a statistical significant decrease during post mating.
- Females (100 mg/kg bw/day) showed a statistical significant decrease during pregnancy and lactation.
Body weight gain was statistically significantly decreased in:
- Males (100 mg/kg bw/day) during the last half of the pre-mating period.
- Females (100 mg/kg bw/day) during pregnancy.
Body weight and body weight gain for males of the 10 and 30 mg/kg dose group were unaffected.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was statistically significantly decreased in:
- Females (100 mg/kg bw/day) during the pre-mating, pregnancy and lactation periods.
Relative food consumption was statistically significantly decreased in:
- Males (100 mg/kg bw/day) during weeks 1, 3 and 5 pre-mating.
- Females (100 mg/kg bw/day) during pre-mating and the first two weeks of pregnancy.
Minor statistically significant differences between controls and 10 or 30 mg/kg bw/d were considered to be of no toxicological relevance.
Details are presented in Table A6.8.2- 2
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The number of implantation sites was statistically significant decreased in dams of the 100 mg/kg bw/day dose group.
The number of living pups at first litter check (day 1 of lactation) was statistically significantly decreased in the 100 mg/kg bw/day dose group.
Total postnatal loss between days 0 and 4 post partum was statistically significantly increased and the viability index was statistically significantly decreased in litters of the 10, 30 and 100 mg/kg bw/day dose groups when compared to the control group. However, these findings were considered to be of no toxicological significance.
Total breeding loss between days 5 and 25 post partum was statistically significantly increased in litters of the 30 mg/kg dose group when compared to the control group. This finding was considered to be of no toxicological significance.
Details are presented in Table A6.8.2- 4.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreases in body weight, body weight gain and relative food consumption in male and female high dose animals (100 mg/kg bw/day); absolute food consumption was significantly decreased in females only
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: P0-dams of the 100 mg/kg bw/d dose group showed a reduced number of implantation sites at necropsy and living pups at day 1 of lactation.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
6 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: decreases in body weight, body weight gain and relative food consumption in male and female high dose animals (20 mg a.i./kg bw/day); absolute food consumption was significantly decreased in females only
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
6 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: P0-dams of the 20 mg a.i./kg bw/d dose group showed a reduced number of implantation sites at necropsy and living pups at day 1 of lactation.
Key result
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical appearance was unaffected when treated up to 100 mg/kg bw/day. An increased incidence in piloerection was noted in animals of the 30 and 100 mg/kg bw/day dose groups during the first two weeks of treatment. A slightly increased incidence in hunched posture was noted in males of the 100 mg/kg dose group during the first two weeks of treatment. Because these findings were transient, they were not considered to be of any toxicological significance. One female of the 30 mg/kg bw/d group showed a palpable mass at the chest from week 15 onwards. This single observation was considered to be caused by chance and thus of no toxicological significance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality was observed at any tested dose level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of males of the 100 mg/kg bw/day dose group were statistically significantly decreased during the pre-mating and mating period.
Females of the 100 mg/kg bw/day dose group showed statistically significantly decreased body weights during the first week of the pre-mating period. Because this was a transient effect, it was not considered to be of toxicological significance. Body weight gain was statistically significantly increased in females of the 10 mg/kg dose group during weeks 3–10 of the pre-mating period. Due to the absence of a dose response relationship, this finding was considered to be of no toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was statistically significantly decreased in males of the 100 mg/kg bw/day dose group during the pre-mating period and the relative food consumption was also slightly lower. During several weeks of the pre mating period, absolute and relative food consumption were statistically significantly decreased in females treated with 100 mg/kg bw/day.
Results are presented in Table A6.8.2- 2.

Females of the 100 mg/kg bw/day dose group showed statistically significantly decreased (relative) food consumption during the last week of pregnancy. Because the mean of means during the pregnancy period was similar as the control group, and no effects were observed during the lactation period, this finding was not considered to be of any toxicological significance. Minor statistically significant differences arising between controls and animals receiving 10 or 30 mg/kg bw/day occurred in the absence of a dose-related response, body weight changes, and/or supportive clinical signs. Therefore, they were considered to be of no toxicological relevance.
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered a result of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Reproduction parameters and breeding data were unaffected by treatment.
Results are presented in Table A6.8.2- 4.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
6 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
20 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Total postnatal loss between days 0 and 4 post partum was statistically significantly increased in litters of the 10, 30 and 100 mg/kg bw/day dose groups when compared to the control group. The increase in postnatal loss in the 10 mg/kg bw/day dose group, was mainly due to three litters in which an increased loss was observed. In one of those litters all fifteen pups died spontaneously or were killed in extremis because the dam died spontaneously due to delivery difficulties. The increase in postnatal loss in the 30 mg/kg bw/day dose group, was mainly due to one litter in which all fourteen pups died on or before day 5 post partum. This was propably due to bad health of the dam (hunched posture and body weight loss). The increase in postnatal loss in the 100 mg/kg bw/day dose group, was mainly due to two litters in which an increased loss was observed. In one of those litters all nine pups died on or before day 5 post partum. This was probably due to bad health of the dam (hunched posture, piloerection, and reduced body weight). Because this statistically significant difference was due to single observations, the number of affected litters was similar for control and treatment groups, and no treatment related change was observed, this finding was considered to be of no toxicological significance.

Total breeding loss between days 5 and 25 post partum was statistically significantly increased in litters of the 30 mg/kg bw/day dose group when compared to the control group. However, this was only a light increase, no dose related response was observed, and it was mainly due to the death of three pups in one litter of which the dam showed bad health. Thus, this finding was considered to be of no toxicological significance.

The viability index was statistically significantly decreased in litters of the 10, 30, and 100 mg/kg bw/day dose groups when compared to the control group. However, because this statistically significant difference was due to single observations, the number of affected litters was similar for control and treatment groups, and not treatment related change was observed, this finding was considered to be of no toxicological significance.
The weaning index was similar for treated and control groups.
Body weight and weight changes:
no effects observed
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
F1-pups
Development of pups was similar for control and treated groups.
No test-item related macroscopic findings were observed upon necropsy of F1-weanlings.
Mean body weights of pups were similar for control and treated groups.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The numbers of dead and living pups per litter at first litter check (day 1 of lactation) were similar for control and treatment groups.

Total postnatal loss between days 0 and 4 post partum was statistically significantly increased in litters of the 10 mg/kg bw/day dose group and statistically significantly decreased in litters of the 100 mg/kg bw/day dose group when compared to the control group. The total number of affected litters by postnatal loss was statistically significantly decreased in litters of the 30 mg/kg bw/day dose group. With a absence of a dose relationsship, this was considered to be caused by chance and to be of no toxicological significance.
Number of affected litters and total breeding loss between days 5 and 25 post partum was statistically significantly increased in litters of the 10 mg/kg bw/day dose group when compared to the control group. As no dose relationship was apparent, this finding was considered to be of no toxicological significance. Total breeding loss between days 5 and 25 post partum was also statistically significantly increased in litters of the 30 mg/kg bw/day dose group. However, no dose related response was observed, and it was mainly due to the death of eight and five pups in two litters. Thus, this finding was considered to be of no toxicological significance.
The viability and weaning inidices were similar for treated and control groups.
Body weight and weight changes:
no effects observed
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
F2-pups
Development of pups was similar for control and treated groups.
No test-item related macroscopic findings were observed upon necropsy of F2-weanlings.
Mean body weights of pups were similar for control and treated groups.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
20 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

 

Table A6.8.2-1:Animal assignment for mating.

 

 

Number of animals

 

 

Control

10 mg/kg /daya

30 mg/kg/daya

100 mg/kg/daya

P mating

Male

24

24

24

24

 

Female

24

24

24

24

F1mating

Male

24

23

23

22

 

Female

24

22

23

22

a: based on test substance as manufactured (20% aqueous solution)

 

Table A6.8.2-2:Observed effects on mortality and body weights during the study.

Parameter

 

 

Control

10 mg/kg/d#

30 mg/kg/d#

100 mg/kg/d#

 

 

Generation

Male

Female

Male

Female

Male

Female

Male

Female

Mortality

 

P

 

 

 

1/24

 

 

2/24

1/24

 

 

F1

 

 

 

 

1/23

2/23

4/22

2/22

Body weight

[g]

 

 

 

 

 

 

 

 

 

Pre-mating

Week 1

Week 3

Week 11

 

P

 

260

336

492

 

272

273

n.a.

 

265

344

504

 

270

270

n.a.

 

266

353

491

 

268

271

n.a.

 

267

341

461

 

264

263

n.a.

Post-mating / Pregnancy

Day 0

Day 1

Day 21

 

 

 

n.a.

494

n.a.

 

277

n.a.

450

 

n.a.

513

n.a.

 

271

n.a.

427

 

n.a.

489

n.a.

 

272

n.a.

433

 

n.a.

452*

n.a.

 

261*

n.a.

397**

Lactation

Day 1

Day 14

Day 25

 

 

 

 

324

360

331

 

 

315

355

326

 

 

316

350

318

 

 

295**

331**

303**

Pre-mating

Week 1

Week 5

Week 10

 

F1

 

117

354

494

 

104

232

284

 

109

353

496

 

96

234

299

 

112

347

478

 

99

235

289

 

102*

323*

428**

 

93*

223

279

Pregnancy

Day 0

Day 21

 

 

 

n.a.

n.a.

 

297

453

 

n.a.

n.a.

 

311

478

 

n.a.

n.a.

 

304

469

 

n.a.

n.a.

 

289

432

Body weight gain

[%]

 

 

 

 

 

 

 

 

 

Pre-mating

Week 6

Week 11

 

P

 

58

89

 

n.a.

n.a.

 

59

90

 

n.a.

n.a.

 

57

84

 

n.a.

n.a.

 

50*

73**

 

n.a.

n.a.

Pregnancy

Day 14

Day 21

 

 

 

 

27

63

 

 

25

58

 

 

25

60

 

 

21**

52**

Pre-mating

Week 6

Week 10

[%]

F1

 

235

329

 

141

177

 

263

362

 

172**

223*

 

245

332

 

154

194

 

241

313

 

154

198

Pregnancy

Day 14

Day 21

 

 

 

 

21

53

 

 

20

54

 

 

21

54

 

 

21

50

n.a.: not applicable

*: Dunnett-test statistically significantly different compared to control (p<0.05); ** (p<0.01)
#:in terms of test substance as manufactured (20% aqueous solution)

 

Table A6.8.2-3:Observed effects on food consumption during the study.

Parameter

 

 

Control

10 mg/kg/d#

30 mg/kg/d#

100 mg/kg/d#

 

 

Generation

Male

Female

Male

Female

Male

Female

Male

Female

Food consumption

[g / animal / day]

 

 

 

 

 

 

 

 

 

Pre–mating

Weeks 1/2

Weeks 2/3

 

P

 

28

28

 

22

21

 

29

30

 

21

20

 

29

30

 

21

20

 

28

28

 

19**

18**

Pregnancy

Days 0–7

Days 7–14

Days 14–21

 

 

 

 

26

27

29

 

 

24*

26

27*

 

 

24*

27

28

 

 

22**

23**

25**

Lactation

Days 1–4

Days 7–14

Days 14–21

 

 

 

 

38

59

71

 

 

33

56

67

 

 

36

56

67

 

 

31**

52**

63*

Pre–mating

Weeks 1/2

Weeks 2/3

Weeks 5/6

Weeks 10/11

[g / animal / day]

F1

 

22

28

35

36

 

18

20

25

24

 

21

27

34

35

 

17

20

26

26

 

19*

25

31

31

 

17

20

24

23

 

18*

23*

28*

27**

 

16**

17*

21*

21

Pregnancy

Days 14–21

 

 

 

 

30

 

 

31

 

 

31

 

 

27*

Relative food consumption

[g / kg bw / day]

 

 

 

 

 

 

 

 

 

Pre–mating

Weeks 1/2

Weeks 2/3

 

P

 

93

85

 

79

77

 

94

88

 

76

74

 

94

85

 

78

75

 

89*

83

 

72**

70**

Pregnancy

Days 0–7

Days 7–14

 

 

 

 

82

78

 

 

79*

76

 

 

80

79

 

 

75**

72*

Pre–mating

Weeks 1/2

Weeks 2/3

[g / kg bw / day]

F1

 

118

111

 

121

111

 

117

110

 

120

110

 

109

102

 

118

107

 

117

103

 

116

98*

Pregnancy

Days 14–21

 

 

 

 

67

 

 

64

 

 

65

 

 

63*

n.a.: not applicable
*: Dunnett-test statistically significantly different compared to control (p<
0.05); ** (p<0.01)
#:
based on test substance as manufactured (20% aqueous solution)

 

 

Table A6.8.2-4:Observed effects on reproductive performance of the two generation study.

 

 

 

Control

10 mg/kg/d a

30 mg/kg/d a

100 mg/kg/d a

Parameter

 

Implantations-sites

Mean

P

15.6

14.3

14.7

13.1+

 

F1

14.0

14.9

15.1

12.6

Living pups at 1. litter check

Mean

P

15.3

13.9

14.2

12.9 +

 

F1

13.8

14.1

14.9

13.0

Postnatal loss days 0-4 p.p.

Total

P

11

41##

21#

25##

 

F1

18

38##

12

4##

Breeding loss days 5–25 p.p.

Total

P

1

3

6+

5

 

F1

0

12##

14##

3

Viability index

 

P

97

87.7##

93.8#

91.6##

 

F1

94.1

87.7

96.3

98.4

+: Steel-test significant at 5% level
# /##: Fisher’s exact test significant at 5% (#) or 1% (##) level
p.p. = post partum
a:
based on test substance as manufactured (20% aqueous solution)

 

 

Conclusions:
In a two generation study, the registration substance (20% a.i., as manufactured) was administered to groups of male and female Wistar rats at 0 (control), 10, 30 or 100 mg/kg bw/day in water, corresponding to 2, 6 and 20 mg a.i./kg bw/day, by gavage during the pre-mating and mating period and to females also during gestation and lactation.
No treatment-related mortality occurred during the study. Parental toxicity consisted of clinical signs (only P0-animals), effects on body weights and food consumption for P0- and P1-animals receiving 100 mg/kg bw/day. Reproductive toxicity consisted of a reduced number of implantation sites at necropsy and a reduced number of living pups on day 1 of lactation for P0-animals receiving 100 mg/kg bw/day. Development of the pups was not affected up to the maximum dose of 100 mg/kg bw/day.
Based on the results of this study, the parental and reproductive NOAEL was established at 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day. The developmental NOAEL was established at 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day.
Executive summary:

The registration substance (20% a.i., as manufactured) was administered to groups of male and female Wistar rats at 0 (control), 10, 30 or 100 mg/kg bw/day inwater, corresponding to 2, 6 and 20 mg a.i./kg bw/day, by gavage during the pre-mating and mating period and to females also during gestation and lactation. The two generation reproduction study was carried out in compliance with OECD guideline 416 (1983) and also with the recent version OECD 416 (2001), except that the examination of oestrus cycle and sperm parameters were not conducted in the study.

 

Parental toxicity

Parental toxicity was assessed by observing mortality, clinical signs, body weights, food consumption, and macroscopic examination for both generations. Mortality and macroscopic examination were unaffected in parental animals P0 and P1-generation up to 100 mg/kg bw/day. At 100 mg/kg bw/day, P0-females showed a slightly increased incidence in hunched posture and P0-animals displayed rales at a low incidence and on few occasions. No treatment-related clinical signs were observed in animals of the P1-generation. P0 - and P1-animals of the 100 mg/kg dose group showed a reduced body weight, body weight gain, food consumption, and relative food consumption during their treatment period. 

Parental NOAEL= 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day based on decreases in body weight, body weight gain and relative food consumption in male and female high dose animals (100 mg/kg bw/day). Absolute food consumption was significantly decreased in females only.

 

Reproductive toxicity

Reproductive toxicity was assessed by observing the number of implantations at birth, mating performance, fertility indices, and breeding data. Mating performance, fertility indices, and breeding data were unaffected in animals of the P0 - and P1-generations when treated up to 100 mg/kg bw/day. P0-dams of the 100 mg/kg bw/day dose group showed a reduced number of implantation sites at necropsy and living pups at day 1 of lactation. The number of implantations at birth was not significant in dams of the P1-generation. 

The increase in postnatal loss in the 10 mg/kg bw/day dose group was due to three litters in which an increased loss was observed. In one of those litters all fifteen pups died spontaneously or were killed in extremis because the dam died spontaneously due to delivery difficulties. The increase in postnatal loss in the 30 mg/kg bw/day dose group was mainly due to one litter in which all fourteen pups died on or before day 5 post partum. This was probably due to bad health of the dam (hunched posture and body weight loss). The increase in postnatal loss in the 100 mg/kg bw/day dose group was mainly due to two litters in which an increased loss was observed. In one of those litters all nine pups died on or before day 5 post partum. This was probably due to poor dam health (hunched posture, piloerection, and reduced body weight). These statistically significant differences were due to single observations, the number of affected litters was similar for control and treatment groups, and no true treatment related effect was observed. There was no dose response associated with these effects. As a consequence of these observations on postnatal loss, the viability index was statistically significantly decreased in litters from all dose groups. These findings are not considered to be toxicologically relevant, they do not display a dose response and as such are not taken into account for determining the reproductive NOAEL. 

Reproductive NOAEL= 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day based on a reduced number (-16% relative to controls) of implantation sites at necropsy of P0 females and the reduced number of living pups at day 1 of lactation.

 

Developmental toxicity

Developmental toxicity was assessed by observing clinical signs, body weights and macroscopic examination of the pups during their lactation period. Clinical signs, body weights and macroscopic examination were unaffected in F1- and F2-pups with maternal treatment up to 100 mg/kg body weight/day. 

Developmental NOAEL= 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day. No treatment-related macroscopic pathological effects were observed for any dose tested. 

 

No treatment-related mortality occurred during the study. Parental toxicity consisted of clinical signs (only P0-animals), effects on body weights and food consumption for P0- and P1-animals receiving 100 mg/kg bw/day. Reproductive toxicity consisted of a reduced number of implantation sites at necropsy and living pups on day 1 of lactation for P0-animals receiving 100 mg/kg bw/day.

 

In conclusion, gavage treatment of male and female Wistar rats with the registration substance (20% a.i.) at dose levels of 10, 30 or 100 mg/kg bw/day, corresponding to 2, 6 and 20 mg a.i./kg bw/day, over two generations revealed parental, and reproductive toxicity in animals receiving 100 mg/kg bw/day. Development of the pups was not affected up to the maximum dose of 100 mg/kg bw/day. 

 

Based on the results of this study, the parental and reproductive NOAEL was established at 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day. The developmental NOAEL was established at 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day.

 

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
6 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.7, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The registration substance (20% a.i., as manufactured) was administered to groups of male and female Wistar rats at 0 (control), 10, 30 or 100 mg/kg bw/day in water, corresponding to 2, 6 and 20 mg a.i./kg bw/day, by gavage during the pre-mating and mating period and to females also during gestation and lactation. The two generation reproduction study was carried out in compliance with OECD guideline 416 (1983) and also with the recent version OECD 416 (2001), except that the examination of oestrus cycle and sperm parameters were not conducted in the study.

Parental toxicity

Parental toxicity was assessed by observing mortality, clinical signs, body weights, food consumption, and macroscopic examination for both generations. Mortality and macroscopic examination were unaffected in parental animals (P) and F1-generation up to 100 mg/kg bw/day. At 100 mg/kg bw/day, P-females showed a slightly increased incidence in hunched posture, while P-animals displayed rales at a low incidence and on few occasions. No treatment-related clinical signs were observed in animals of the F1-generation. P- and F1-animals of the 100 mg/kg dose group showed a reduced body weight, body weight gain, food consumption, and relative food consumption during their treatment period. 

Parental NOAEL= 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day based on decreases in body weight, body weight gain and relative food consumption in male and female high dose animals (100 mg/kg bw/day). Absolute food consumption was significantly decreased in females only.

 

Reproductive toxicity

Reproductive toxicity was assessed by observing the number of implantations at birth, mating performance, fertility indices, and breeding data. Mating performance, fertility indices, and breeding data were unaffected in animals of the P0 - and P1-generations when treated up to 100 mg/kg bw/day. P0-dams of the 100 mg/kg bw/day dose group showed a reduced number of implantation sites at necropsy and living pups at day 1 of lactation. The number of implantations at birth was not significant in dams of the P1-generation. 

The increase in postnatal loss in the 10 mg/kg bw/day dose group was due to three litters in which an increased loss was observed. In one of those litters all fifteen pups died spontaneously or were killed in extremis because the dam died spontaneously due to delivery difficulties. The increase in postnatal loss in the 30 mg/kg bw/day dose group was mainly due to one litter in which all fourteen pups died on or before day 5 post partum. This was probably due to bad health of the dam (hunched posture and body weight loss). The increase in postnatal loss in the 100 mg/kg bw/day dose group was mainly due to two litters in which an increased loss was observed. In one of those litters all nine pups died on or before day 5 post partum. This was probably due to poor dam health (hunched posture, piloerection, and reduced body weight). These statistically significant differences were due to single observations, the number of affected litters was similar for control and treatment groups, and no true treatment related effect was observed. There was no dose response associated with these effects. As a consequence of these observations on postnatal loss, the viability index was statistically significantly decreased in litters from all dose groups. These findings are not considered to be toxicologically relevant, they do not display a dose response and as such are not taken into account for determining the reproductive NOAEL. 

Reproductive NOAEL = 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day based on a reduced number (-16% relative to controls) of implantation sites at necropsy of P0 females and the reduced number of living pups on day 1 of lactation.

 

Developmental toxicity

Developmental toxicity was assessed by observing clinical signs, body weights and macroscopic examination of the pups during their lactation period. Clinical signs, body weights and macroscopic examination were unaffected in F1- and F2-pups with maternal treatment up to 100 mg/kg bw/day. 

Developmental NOAEL= 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day. No treatment-related macroscopic pathological effects were observed for any dose tested. 

 

No treatment-related mortality occurred during the study. Parental toxicity consisted of clinical signs (only P0-animals), effects on body weights and food consumption for P0- and P1-animals receiving 100 mg/kg bw/day. Reproductive toxicity consisted of a reduced number of implantation sites at necropsy and living pups on day 1 of lactation for P0-animals receiving 100 mg/kg bw/day.

In conclusion, gavage treatment of male and female Wistar rats with the registration substance (20% a.i.) at dose levels of 10, 30 or 100 mg/kg bw/day, corresponding to 2, 6 and 20 mg a.i./kg bw/day, over two generations revealed parental, and reproductive toxicity in animals receiving 100 mg/kg bw/day. Development of the pups was not affected up to the maximum dose of 100 mg/kg bw/day. 

Based on the results of this study, the parental and reproductive NOAEL was established at 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day. The developmental NOAEL was established at 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day.


Effects on developmental toxicity

Description of key information

- prenatal developmental toxicity study; oral(gavage), rabbit (New Zealand White, 15 females/dose), OECD TG 414, GLP;
NOAEL(maternal) = 10 mg a.i./kg bw/d, based on decrease in body weight gain and food consumption at 30 mg a.i./kg bw/d
NOAEL(development) = 30 mg a.i./kg bw/d, no foetal or reproductive toxicity
- prenatal developmental toxicity study; oral(gavage), rat (Wistar rats Crl: (WI) BR, 24 females/dose), OECD TG 414, GLP;
NOAEL(maternal) = 20 mg a.i./kg bw/d, based on clinical signs, reduced body weight gain and food consumption at 50 mg a.i./kg bw/d
NOAEL(development) = 50 mg a.i./kg bw/d, no foetal or reproductive toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1981)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar rats Crl: (WI) BR (outbred, SPF-Quality)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: Approx. 10 weeks
- Weight at study initiation: 221–310 g
- Housing: in groups of 5 during acclimatisation, durung mating females were caged together with males on a one-to-one-basis; individually during study
- Diet (e.g. ad libitum): standard pelleted laboratory animal diet (from Car-51 Quality BVBA, Oud-Turnhout, Belgium), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 12 d prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-100
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulations revealed values for accuracy within the range of 99% and 103% of nominal for the week 1 formulations, and between 88% and 119% of nominal for the week 3 formulations. This was considered to represent an acceptable level for formulations of this type.
The low and high dose formulations were considered to be stable over 4 hours and sufficiently homogenous.
Method of analysis: TOC analysis; this is considered suitable in view of the fact that the test substance was the only organic constituent in the dosing solutions.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Once mating had occurred, the female was separated from the male and vaginal smearing was discontinued. When sufficient mated females were obtained for each dose group, the surplus females were removed from the study.

Duration of treatment / exposure:
Day 6–16 of gestation
Frequency of treatment:
daily
Duration of test:
21 d
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
based on test material (20% formulation)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
based on test material (20% formulation)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
based on test material (20% formulation)
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for the present embryotoxicity and teratogencity study were based on a preliminary study in pregnant rats.
Maternal examinations:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- mortality/viability: twice daily

BODY WEIGHT: Yes
On day 0 and 3, daily from day 6 to day 17 inclusive and on day 21 post-coitum

FOOD CONSUMPTION: Yes
during days 0–3, 3–6, 6–9, 9–13, 13–17 and 17–21 post-coitum

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
ovaries and uterine content
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data can not be assumed to follow a normal distribution.
The exact Fisher-test was applied for 2x2 tables if variables could be dichotimized without loss of information.
The Student’s t test was applied on placenta data to test the difference between the treatment and the control groups.
All tests will be two-sided and in all cases p < 0.05 were accepted as the lowest level of significance.
Indices:
Implantation index, Implantation site scar index, Embryonic/foetal death index, Embryonic resorption index, Foetal resorption index, Total foetus index, Live foetus index, Dead foetus index, Abnormal foetus index, Percentage males, Percentage females, Percentage live males, Percentage live females
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Four animals treated with 250 mg/kg bw/day showed incidental findings (hunched posture, laboured respiration, rales, brown discolouration of the snout, salivation and piloerection) which were however considered to be related to treatment. One animal treated with 40 mg/kg bw/day showed scabs on the nose and one which received 100 mg/kg bw/day showed piloerection. These effects were considered not to be treatment related. Alopecia was observed in all animals of all dose groups. Alopecia is commonly seen in animals of this strain used in this type of study and therefore no toxicological significance was attached to this finding.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females receiving 250 mg/kg bw/day showed a decrease in body weight and body weight gain, which was statistically significant on gestation days 13 to 17 and 11 to 17, when compared to the control group. After correction of the uterus weight, a decrease in body weight gain was noted in the 250 mg/kg bw/day dose group when compared to the control. However, this was not statistically significant due to the high standard deviation in this treated group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females treated with 250 mg/kg bw/day showed a statistically significant decrease in food consumption on gestation days 6 to 17. This finding was considered to be caused by the test substance. Females receiving 100 mg/kg bw/day showed a slight, statistically significant decrease in food consumption from gestation days 6–9 and 13–17 when compared to the control. However, this difference was considered to be unrelated to treatment because the mean of relative food consumption of females of this dose group were comparable with the control values.
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related macroscopic lesions were observed upon necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant differences seen in pre-implantation loss and implantation index of the 40 and 250 mg/kg bw/d dose group were considered to be unrelated to treatment, since treatment started on gestation day 6, which is after implantation. No treatment related effects were seen on post-implantation loss.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead foetus was noted in the 40 mg/kg bw/day dose group upon caesarean sectioning. The number of live foetuses observed in the treatment and control groups remained within biologically normal limits for rats of this age and strain and no treatment related differences were observed.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
With the exception of one female of the 100 mg/kg bw/day dose group, all females were pregnant. The non-pregnant female was excluded from further calculations.
Other effects:
not examined
Details on maternal toxic effects:
Results are presented in Table A6.8.1- 5 and Table A6.8.1- 6.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Foetal weights and of dams and placental weights of live foetuses of the 40 mg/kg bw/d group were significantly decreased when compared to the control group. Due to absence of a dose-relation, this change was considered to be unrelated to treatment.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The number of live foetuses observed in the treatment and control groups remained within biologically normal limits for rats of this age and strain and no treatment related differences were observed.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
External examination of the foetuses did not reveal any findings among foetuses of litters treated with the test substance that were considered to be an adverse effect of the test substance. There are minor morphological changes amongst all litters across all dose groups as well as the control. In general there were no indications of any consistent treatment or dose related effects on any of the parameters investigated, some of which are tabulated in A6.8.1-4 for illustrative purposes. Malrotated limbs, and small foetuses were noted in all groups. Incidental findings consisted of two very
autolysed foetuses (of which one foetus was dead), and an umbilical hernia
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Compared with the concurrent controls and the 40 mg/kg bw/day dose group, there appeared to be indications of a very marginal reduction in ossification in the 250 mg/kg bw/day dose group. Parameters affected included a number of cranial bones, viz. supraoccipital, interparietal, squamosal, jugal, maxilla(e) and hyoid, the caudal vertebral arches and humerus(i). Other ossification parameters were similar in all groups.
However, reference to laboratory historical control data indicated that the concurrent control values showed a slightly advanced ossification, whilst the majority of the values in the 100 and 250 mgkg bw/day dose groups were within the historical control ranges.
It was considered, therefore, that the slight differences from the concurrent controls were of no toxicological significance.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A number of anomalies were recorded in all groups, the types, incidences and group distribution of which did not indicate any association with treatment.
In the 100 and 250 mg/kg bw/day dose groups there appeared to be a marginal increase in the incidence of unilateral hydroureter compared with the concurrent controls and the 40 mg/kg bw/day dose group, however, as no increases were recorded in the incidences of bilateral hydroureter or renal pelvic cavitation it was considered that this finding did not represent a toxicologically significant response to treatment.
In the 250 mg/kg bw/day dose group one litter contained four foetuses with an accessory blood vessel associated with the superior pole of the kidney and a second litter contained one small foetus with cranial abnormalities and immature gonads. The isolated nature of these findings did not suggest any association with treatment.
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table A6.8.1-3: Rat Maternal and Foetal Observations made at necropsy.

Parameter

Control

40 mg/kg/d

100 mg/kg/d

250 mg/kg/d

Number of dams examined

24

24

23a

24

Mortality of dams

0

0

0

0

Mean body weight [g] at day 0

262

267

256

261

Mean body weight [g] at day 17

364

377

352

341**

Mean body weight [g] at day 21

439

455

423

418

Mean body weight gain [%] day12

23

24

23

19**

Mean body weight gain [%] day 17

39

41

38

31**

Mean food consumption [g/rat/day], day 6–9

27

28

25*

23**

Mean food consumption [g/rat/day], day 9–13

28

28

26

22**

Mean food consumption [g/rat/day], day 13–17

29

30

26*

23**

Relative food consumption [g/kg bw/day], day 13–17

85

86

80

70**

Number of pregnant females

24/24

24/24

23/24

24/24

Abortions

0

0

0

0

 

 

 

 

 

No. of litters totally resorbed

0

0

0

0

No. of litters with viable foetuses

24

24

23

24

No. of corpora lutea/dam

18.6 ± 2.8

19.7 ± 2.9

17.9 ± 2.8

17.8 ± 2.1

No. of implantations/dam

15.5 ± 2.9

17.3 ± 2.4

15.0 ± 2.8

16.0 ± 2.6

Mean% pre-implantation loss

16.6

11.9#

16.5

10.1##

No. of resorptions/litter

0.5

0.5

0.6

0.9

Mean% post-implantation loss

3.5

2.9

4.1

5.5

Viable foetuses (total)

359

404

330

363

Viable foetuses/litter

15.0 ± 2.9

16.8 ± 2.3

14.3 ± 3.0

15.1 ± 3.2

Dead foetuses (total)

0

1

0

0

Foetal weight-males (g)

5.3 ± 0.5

5.3 ± 0.6

5.4 ± 0.4

5.4 ± 0.5

Foetal weight-females (g)

5.0 ± 0.5

4.9 ± 0.8

5.1 ± 0.4

5.1 ± 0.5

Foetal weight-sexes combined (g)

5.2 ± 0.5

5.1 ± 0.7

5.2 ± 0.4

5.2 ± 0.5

Sex ratio (M%:F%)

48:52

48:52

50:50

52:48

Mean placental weight (g)

0.51 ± 0.06

0.47 ± 0.06

0.53 ± 0.06

0.50 ± 0.06

 

 

 

 

 

a) one animal (female 51) did not become pregnant after mating.

* : Dunnett-test based on pooled variance significant at 5% level

** : Dunnett-test based on pooled variance significant at 1% level

#: Fisher’s Exact Test significant at level 5%

##: Fisher’s Exact Test significant at level 1%

 

Table A6.8.1-4: Incidence of Selected Rat Foetal Parameters*(Caesarean section data from day 21).

Parameter

Control

40 mg/kg/d

100 mg/kg/d

250 mg/kg/d

 

 

Number of foetuses (no. of litters) examined

visceral examination

182 (24)

201 (24)

162 (23)

180 (24)

 

skeletal examination

185 (24)

202 (24)

168 (23)

183 (24)

 

Visceral Examination - affected foetuses (no. of litters)

 

small additional vessel arising from aorta

1 (1)

0 (0)

0 (0)

0

 

small additional liver lobe

14 (12)

12 (9)

14 (7)

14 (11)

 

kidney and adrenal gland displaced

7 (6)

9(5)

4(4)

5(5)

 

↑ renal pelvic cavitation: unilateral

0 (0)

0 (0)

1 (1)

2 (2)

 

↑ renal pelvic cavitation: bilateral

1(1)

0 (0)

0 (0)

0 (0)

 

hydroureter: unilateral
           % of total foetuses:

4 (3)
2.2

5 (4)
2.5

12 (9)
7.1

17 (9)
9.3

 

hydroureter: bilateral

4 (2)

4 (3)

0 (0)

6 (5)

 

Skeletal Examination - affected foetuses (no. of litters)

 

delayed ossification interparietal
           % of total foetuses:

23 (13)
12.4

23 (13)
11.4

31 (10)
18.5

42 (13)
23.0

 

delayed ossification supraoccipital
           % of total foetuses:

4 (2)
2.2

7 (5)
3.5

12 (7)
7.1

19 (10)
10.4

 

delayed ossification hyoid

2 (2)

1 (1)

8 (6)

6 (3)

 

delayed ossification sternebrae (≥3)

2 (2)

3 (3)

1 (1)

6 (5)

 

anomalous sternabrae

9 (6)

7 (7)

7 (7)

14 (11)

 

Head Examination – affected foetuses (no. of litters)

 

bilateral slightly folded retina

1 (1)

0 (0)

0 (0)

1 (1)

 

 

 

 

 

 

 

*Some of the effects seen multiple times at any dose are recorded here. There was no consistent relationship between dose and any of the recorded effects noted in the original pathologist‘s report except for the incidence of hydroureter. Numbers in parenthesis indicate the number of litters in which that effect is found. 

 

 

Visceral Examination

A number of anomalies were recorded in all groups, the types, incidences and group distribution of which did not indicate any association with treatment.

In the intermediate and high dose groups (100 and 250 mg/kg body weight/day) there appeared to be a marginal increase in the incidence of unilateral hydroureter compared with the concurrent controls and the low dose group (40 mg/kg body weight/day), however, as no increases were recorded in the incidences of bilateral hydroureter or renal pelvic cavitation it was considered that this finding did not represent a toxicologically significant response to treatment.

In the high dose group (250 mg/kg body weight/day) one litter contained four fetuses with an accessory blood vessel associated with the superior pole of the kidney and a second litter contained one small foetus with cranial abnormalities and immature gonads. The isolated nature of these observations did not suggest any association with treatment.

 

Skeletal Examination

Compared with the concurrent controls and the low dose group (40 mg/kg body weight/day), there appeared to be indications of reduction in foetal ossification in the high dose group (250 mg/kg body weight/day) and, to a lesser extent, in the intermediate dose group (100 mg/kg body weight/day). Parameters affected included a number of cranial bones, viz. supraoccipital, interparietal, squamosal, jugal. maxilla(e) and hyoid, the caudal vertebral arches and humerus(i). Other ossification parameters were similar in all groups. However, reference to laboratory historical control data indicated that the concurrent control values showed a slightly advanced ossification, whilst the majority of the values in the 100 and 250 mgkg dose groups were within the historical control ranges. It was considered, therefore, that the slight differences from the concurrent controls were of no toxicological significance.

 

Morphological parameters

A number of foetuses in all groups showed minor morphological changes, but there were no indications of any consistent treatment or dose related associations or trends.

Conclusions:
Dams treated with 250 mg/kg bw/day registration substance (20% a.i.) showed maternal toxicity comprising a decrease in body weight and body weight gain, which were statistically significant on gestation days 13 to 17 and 11 to 17, when compared to the control group. Females treated with 250 mg/kg bw/day showed a statistically significant decrease in food consumption on gestation days 6 to 17. Four animals out of 24 in the high dose group showed generalised clinical findings including hunched posture, laboured respiration, rales, brown discolouration of the snout, salivation and piloerection. The maternal NO(A)EL is set at 100 mg/kg bw/day (corresponding to 20 mg a.i. /kg bw/day).

External, visceral, skeletal examination of the foetuses did not reveal any findings among foetuses of litters treated with the test substance that were considered to be an adverse effect of the test substance. Oral administration of the registration substance (20% a.i.) to pregnant Wistar rats during the period of organogenesis at dose levels up to 250 mg/kg bw/day (corresponding to 50 mg a.i./kg bw/day) did not result in any adverse effects during foetal development.
Executive summary:

The potential of the registration substance (20% a.i.) to induce embryotoxic effects in the rat (Wistar rats Crl: (WI) BR (outbred, SPF-Quality)) was investigated at 0 (control), 40, 100 and 250 mg/kg bw/day in water, administered from day 6 to day 16 post coitum, according to OECD guideline 414 (1981). Furthermore, the conduct of the study was consistent to the recent OECD guideline 414 (2001) in all important aspects, except that test substance was administered solely during the period of organogenesis.

 

Visceral Examination

A number of anomalies were recorded in all groups, the types, incidences and group distribution of which did not indicate any association with treatment.

In the intermediate and high dose groups (100 and 250 mg/kg body weight/day) there appeared to be an increase in the incidence of unilateral hydroureter compared with the concurrent controls and the low dose group (40 mg/kg body weight/day), however, as no increases were recorded in the incidences of bilateral hydroureter or renal pelvic cavitation it was considered that this finding did not represent a toxicologically significant response to treatment.

In the high dose group (250 mg/kg body weight/day) one litter contained four fetuses with an accessory blood vessel associated with the superior pole of the kidney and a second litter contained one small foetus with cranial abnormalities and immature gonads. The isolated nature of these observations did not suggest any association with treatment.

 

Skeletal Examination

Compared with the concurrent controls and the low dose group (40 mg/kg body weight/day), there appeared to be indications of a reduction in foetal ossification in the high dose group (250 mg/kg body weight/day) and, to a lesser extent, in the intermediate dose group (100 mg/kg body weight/day). Parameters affected included a number of cranial bones, viz. supraoccipital, interparietal, squamosal, jugal. maxilla(e) and hyoid, the caudal vertebral arches and humerus(i). Other ossification parameters were similar in all groups.

 

Morphological Examination

A number of foetuses in all groups showed minor morphological changes, but there were no indications of any consistent treatment or dose related associations or trends.

 

Dams treated with 250 mg/kg bw/d showed maternal toxicity comprising clinical signs, reduced body weight gain and food consumption during the treatment period. Based on the results of this study, DOPA-Glycinate (20% a.i.) is considered not to be teratogenic when orally administered to Wistar rats at levels up to and including 40 mg/kg bw/d.

The maternal no observed adverse effect level (NOAEL) was 100 mg/kg bw/d, corresponding to 20 mg a.i./kg bw/d.

The NOAEL for survival, growth and development in utero was 40 mg/kg bw/d, corresponding to 8 mg a.i./kg bw/d.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-07-05 to 2000-10-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1981)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Germany, Kißlegg, Germany
- Weight at study initiation: 3490 g
- Housing: individually in cages with perforated floors
- Diet: ad libitum
- Water:ad libitum
- Acclimation period: 5 weeks prior to allocation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance formulations in water formed a homogeneous solution (the test report states “suspension”, which is, however, unlikely in view of the nature of the test item) at the concentrations tested (concentrations were 91–105% of nominal).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the accuracy of formulations on the 2nd day of administration revealed values of 121 to 135% of nominal concentration, which exceeded the acceptable range of 100±10%. However, these measurements were not representative for the whole treatment period. Towards the end of the dosing period analysis gave results between 98 and 106% of nominal concentrations. Method of analysis: TOC analysis; this is considered suitable in view of the fact that TEGO 2000 was the only organic constituent in the dosing solutions.
Details on mating procedure:
- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: yes
Three weeks prior to insemination all female rabbits received an initial intramuscular injection of 0.2 ml Receptal (Hoechst, Holland N.V., Amsterdam, The Netherlands) into the muscles of one hind leg. The females were fertilized by artificial insemination with 1 ml diluted sperm sample (diluted with Betsville F5 (BF5) medium). The sperm was collected from donor bucks of the same strain and source and microscopically checked for viability, motility and impurity. The female rabbits were inseminated the same morning as sperm collection. Immediately after insemination again 0.2 ml Receptal (Hoechst) was injected into the muscles of a hind leg of each animal. This day was designated day 0 post coitum.
Duration of treatment / exposure:
Day 6–18 post-coitum
Frequency of treatment:
Once daily from day 6 to day 18 post coitum inclusive, approximately the same time each day
Duration of test:
Duration of exposure Day 6–18 post-coitum
Post-exposure period Day 19–28 post-coitum
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
based on test material (20% formulation)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
based on test material (20% formulation)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
based on test material (20% formulation)
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on a dose range finding study in unmated rabbits (294323) with TMC125K (HBr) in which 500, 750, and 1000 mg/kg body weight/day were administered. All animals died prior to scheduled necropsy All animals of the 750 mg/kg and 1000 mg/kg dose groups showed corrosiveness of the test substance on the stomach. Because the 750 and 1000 mgkg dose levels were obviously too high and no effects of the test substance were observed in the 500 mg/kg dose group, 50,150 and 450 mg/kg were chosen as the dose levels for the pilot teratology rabbit study. All animals dosed at 450 mg/kg bw/day died, therefore lower dose levels (0, 15, 50 and 150 mg/kg bw/day) were selected for the main study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from day 0 post cotium onwards.
Cage debriswas examined to detect abortion or premature birth.

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 and 3, daily from day 6 to day 19 inclusive and on days 22, 25 and 28 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

- Time schedule for examinations: During days 0–3, 3–6, 6–9, 9–12, 12–15, 15–19, 19–22, 22–25, and 25–28 post-coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 28
- Organs examined: ovaies, uteri, foetuses
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyse the data: If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups. The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data can not be assumed to follow a normal distribution. The exact Fisher-test was applied for 2x2 tables if variables could be dichotimized without loss of information The Student’s t test was applied on placenta data to test the difference between the treatment and the control groups. All tests will be two-sided and in all cases p < 0.05 were accepted as the lowest level of significance.
Indices:
Embryonic resorption index, Foetal resorption index, Total foetus index, Live foetus index, Dead foetus index, Abnormal foetus index, Percentage males, Percentage females, Percentage live males, Percentage live females
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Five animals of the high dose group showed lethargy during treatment for one or several days.
The animal that was killed in extremis showed lethargy, pale appearance, diarrhoea, emaciation and hypothermia during treatment. These findings were considered to be related to treatment. One animal in this dose group showed red staining of the genital region for one day during pretreatment. No treatment related effects were observed in rabbits of the other dose groups. At 50 mg/kg, one animal showed absence of faeces on one day after the treatment period, and one animal showed focal etythema and alopecia on the right hindleg. One animal of the control group showed lethargy on one day during treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One female of the high dose group was sacrificed non-scheduled after showing severe clinical signs. One animal of the mid dose group was sacrificed non-scheduled after an early delivery. Another animal of the low dose group died incidental (intubation error).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females receiving 150 mg/kg bw/day showed a decrease in body weight gain, which was statistically significant on gestation days 15 and 17 to 22, when compared to the control group. These findings were considered to be caused by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption and relative food consumption of females treated with 150 mg/kg b.w./day were decreased when compared to the control group, and showed a statistically significant decrease on gestation days 15 to 19. This finding was considered to be caused by treatment of the test substance. Food consumption and relative food consumption of females treated with 50 mg/kg bw/day were decreased when compared to the control group, however this was not statistically significant. Since these changes occurred in the absence of clinical signs or effects on body weight (gain) in this group, they were considered to be of no toxicological relevance.
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any lesions which were considered to be related to treatment.
One female of the control group showed a spleen with irregular surface, and one control female showed ovaries that were reduced in size. One female of the 15 mg/kg group had many grey/white foci on the gallbladder, and one female of the 50 mg/kg bw/day dose group showed many dark red foci on the lungs.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
With the exception of two females of the control group, one of the 50 mg/kg bw/day group and one of the 150 mg/kg bw/day group, all females surviving to day 28 post-coitum were pregnant. It could not be established if the female of the 15 mg/kg bw/day which was found dead and the female of the 150 mg/kg bw/day dose group which was killed in extremis, were pregnant. Two females of the control group showed implantation sites only, and one female of the 50 mg/kg dose group showed an early delivery. These females were excluded from further calculations.
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Foetal body weights were considered not to have been affected by treatment with the test substance.
Foetal weights of dams receiving 50 mg/kg bw/day were statistically significantly decreased on individual basis when compared to the control group. This finding was not dose-related and was considered to be caused by a higher number of foetuses per litter in this dose group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Litter sizes were considered not to have been affected by treatment with the test substance.
Three dead foetuses were noted (two in the 50 mg/kg bw/day group, and one in the 150 mg/kg bw/day group) upon caesarean sectioning. The number of live foetuses observed in the treatment and control groups remained within biologically normal limits for rabbits of this age and strain and no treatment related differences were observed.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no findings that were considered to be related to maternal treatment at any level.
External examination of the foetuses revealed limb malrotations and small foetuses. The incidence of these findings were similar for treated as control groups. Incidental findings consisted of two foetuses of one dam of the 50 mg/kg bw/day dose group which were dead and very small at necropsy. This finding was considered to be caused by chance and not to be of any toxicological significance.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no consistent dose or treatment related associations or trends that were considered to represent an adverse response in ossification parameters to treatment with the registration substance.
A number of foetuses in all groups showed minor morphological changes, but there were no indications of any consistent treatment or dose related associations or trends.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no findings that were considered to be related to maternal treatment at any level.
Visceral examination revealed a small number of anomalies in all groups, including the control group.
Major findings were observed in a few foetuses. One foetus of the control group showed a thoracic cavity filled with fluid, lungs very reduced in size, tricuspid atresia, pulmonary atresia, pulmonary arteries supplied by reflux via ductus arteriosus, right ventricle very small, and left ventricle enlarged. One foetus of the 15 mg/kg bw/day group showed pulmonary stenosis, enlarged left ventricle, reduced right ventricle, and tricuspid stenosis, and one foetus of this dose group showed a diafragmatic hernia on the left side stomach, and a small left lung as a result of this. One foetus of the 50 mg/kg bw/day showed a cyste associated with median lung lobe which was filled with clear fluid, and two foetuses of this dose group showed an inter-ventricular septal defect. One foetus of the 150 mg/kg bw/day dose group, showed an interventricular septal defect.
All-findings occurred in the absence of a dose-response relationship and the findings were considered to be within the normal range of background alterations that may be seen in untreated animals of this age and strain.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue examination of foetal heads:
No gross changes were seen in any of the foetal heads from any of the dose levels tested.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Maternal effects

Parameter

Control

15 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Dose response
+/–

Number of dams examined

15

15

15

15

 

Mortality of dams

0

1

1

1

 

Clinical findings (lethargy)

1

0

0

6a

+

Mean body weight [g] at day 0

3499

3516

3436

3508

 

Mean body weight [g] at day 28

4113

4021

3951

3982

 

Mean body weight gain [%] at day15

11

11

11

8*

Mean body weight gain [%] at day 18

13

12

12

8**

Mean food consumption [g/rabbit/day], day 0–3

176

171

158

172

 

Mean food consumption [g/rabbit/day], day 15–19

164

162

143

111*

Mean food consumption [g/rabbit/day], day 25–28

99

73

81

82

 

Number of pregnant females

13/15

14/15

13/15

13/15

 

Abortions

2

(implantation sites only)

1

(early delivery)

0

0

 

a) one animal which was killed in extremis showed pale appearance, diarrhoea, emaciation and hypothermia

* : Dunnett-test based on pooled variance significant at 5% level

** : Dunnett-test based on pooled variance significant at 1% level

 

 

Litter data of dams with live young at day 29 (Caesarean section data)

Parameter

Control

15 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Dose response
+/–

Fetuses
(dead/total )

0/105

0/123

2/136

1/116

 

Sex ratio
(male/female)

57/48

55/68

77/57

72/43

 

Mean foetal body weights [g]

34.6

34.0

31.8

34.5

 

 

 

Conclusions:
Based on the results of this study, the registration substance (20% a.i.) was considered not to be teratogenic when orally administered to New Zealand White rabbits at levels up to and including 150 mg/kg bw/day, corresponding to 30 mg a.i./kg bw/day.
Executive summary:

The effects of the registration substance (20% a.i.) on the pregnancy and embryonic or foetal development of the rabbit were investigated at 0 (control), 15, 50 and 150 mg/kg bw/day administered in water from day 6 to day 18 post mating.

The study was performed according to OECD 414 (1981). The conduct of the study is consistent in all important aspects to OECD 414 (2001), except for the number of rabbits (15 per group) which is, however, conform to OECD guideline 414 (1981), the version still effective at the time of conduct of the study. The test substance was administered solely during the period of organogenesis.

There was no maternal or developmental toxicity associated with oral administration at 15 or 50 mg/kg bw/day. Dams treated with 150 mg/kg bw/day showed maternal toxicity characterised by mortality, clinical signs, reduced body weight gain and food consumption during the treatment period. No foetal or reproductive toxicity was observed at these dose levels.

One female of the high dose group was sacrificed non-scheduled after showing severe clinical signs. One animal of the mid dose group was sacrificed non-scheduled after an early delivery. Another animal of the low dose group died incidental (intubation error).

Five animals of the high dose group showed lethargy during treatment for one or several days. The sacrificed animal (see above) showed lethargy, pale appearance, diarrhoea, emaciation and hypothermia during treatment. These findings were considered to be related to treatment.

No treatment related effects were observed in rabbits of the mid dose group.

Animals of the low dose group showed no clinical signs.

Females receiving 150 mg/kg bw/day showed a decrease in body weight gain, which was statistically significant on gestation days 15 and 17 to 22, when compared to the control group. These findings were considered to be caused by treatment with the test item.

Females treated with 150 mg/kg bw/day showed a statistically significant decrease in food consumption on gestation days 15 to 19. This finding was considered to be caused by treatment of the test substance.

No treatment-related macroscopic lesions were observed upon necropsy.

No treatment related differences in reproduction parameters were observed.

No treatment-related effects were observed on the litter size. Three dead foetuses (two in the 50 mg/kg bw/d group; one in the 150 mg/kg bw/d group) remained within biologically normal limits for rabbits of this age and strain.

No treatment-related differences were observed in sex ratio.

Foetal body weights were considered to have not been affected by treatment with the test substance. Also foetal weights of dams treated with 50 mg/kg bw/d showed a statistical significantly decrease compared to the control; this effect was not dose-related and considered to be caused by a higher number of foetuses per litter.

The placental weights of live foetuses of the treated and control groups were similar.

There were no treatment-related effects on the nature or incidence of major or minor external, visceral or skeletal anomalies.

Soft tissue examination of foetal heads showed a slightly lower incidence of foetuses with eruption of the lower incisors in the treated group than in the control group. However, no dose related trend was apparent and this effect was not considered to represent a toxicologically significant response to treatment.

Based on the results of this study, the registration substance (20% a.i.) was considered not to be teratogenic when orally administered to New Zealand White rabbits at levels up to and including 150 mg/kg bw/day, corresponding to 30 mg a.i./kg bw/d.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.7, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

For the assessment of developmental toxicity of the registration substance prenatal developmental toxicity studies in rabbits and one in rats is available. Furthermore, data from a two generation study are available as well as described above.

 

Study in rabbits

The effects of the registration substance (20% a.i.) on the pregnancy and embryonic or foetal development of the rabbit were investigated at 0 (control), 15, 50 and 150 mg/kg bw/day administered in water from day 6 to day 18 post mating.

The study was performed according to OECD 414 (1981). The conduct of the study is consistent in all important aspects to OECD 414 (2001), except for the number of rabbits (15 per group) which is, however, conform to OECD guideline 414 (1981), the version still effective at the time of conduct of the study. The test substance was administered solely during the period of organogenesis.

There was no maternal or developmental toxicity associated with oral administration at 15 or 50 mg/kg bw/day. Dams treated with 150 mg/kg bw/day showed maternal toxicity characterised by clinical signs, reduced body weight gain and food consumption during the treatment period. No foetal or reproductive toxicity was observed at these dose levels.

One female of the high dose group was sacrificed non-scheduled after showing severe clinical signs. One animal of the mid dose group was sacrificed non-scheduled after an early delivery. Another animal of the low dose group died incidental (intubation error).

Five animals of the high dose group showed lethargy during treatment for one or several days. The sacrificed animal (see above) showed lethargy, pale appearance, diarrhoea, emaciation and hypothermia during treatment. These findings were considered to be related to treatment.

No treatment related effects were observed in rabbits of the mid dose group.

Animals of the low dose group showed no clinical signs.

Females receiving 150 mg/kg bw/day showed a decrease in body weight gain, which was statistically significant on gestation days 15 and 17 to 22, when compared to the control group. These findings were considered to be caused by treatment with the test item.

Females treated with 150 mg/kg bw/day showed a statistically significant decrease in food consumption on gestation days 15 to 19. This finding was considered to be caused by treatment of the test substance.

No treatment-related macroscopic lesions were observed upon necropsy.

No treatment related differences in reproduction parameters were observed.

No treatment-related effects were observed on the litter size. Three dead foetuses (two in the 50 mg/kg bw/day group; one in the 150 mg/kg bw/day group) remained within biologically normal limits for rabbits of this age and strain.

No treatment-related differences were observed in sex ratio.

Foetal body weights were considered to have not been affected by treatment with the test substance. Also foetal weights of dams treated with 50 mg/kg bw/day showed a statistical significantly decrease compared to the control; this effect was not dose-related and considered to be caused by a higher number of foetuses per litter.

The placental weights of live foetuses of the treated and control groups were similar.

There were no treatment-related effects on the nature or incidence of major or minor external, visceral or skeletal anomalies.

Soft tissue examination of foetal heads showed a slightly lower incidence of foetuses with eruption of the lower incisors in the treated group than in the control group. However, no dose related trend was apparent and this effect was not considered to represent a toxicologically significant response to treatment.

Based on the results of this study, the registration substance (20% a.i.) was considered not to be teratogenic when orally administered to New Zealand White rabbits at levels up to and including 150 mg/kg bw/day, corresponding to 30 mg a.i./kg bw/day.

 

Study in rats

The potential of registration substance (20% a.i.) to induce embryotoxic effects in the rat (Wistar rats Crl: (WI) BR (outbred, SPF-Quality)) was investigated at 0 (control), 40, 100 and 250 mg/kg bw/day in water, administered from day 6 to day 16 post coitum, according to OECD guideline 414 (1981). Furthermore, the conduct of the study was consistent to the recent OECD guideline 414 (2001) in all important aspects, except that test substance was administered solely during the period of organogenesis.

Visceral Examination

A number of anomalies were recorded in all groups, the types, incidences and group distribution of which did not indicate any association with treatment.

In the intermediate and high dose groups (100 and 250 mg/kg body weight/day) there appeared to be an increase in the incidence of unilateral hydroureter compared with the concurrent controls and the low dose group (40 mg/kg body weight/day).

In the high dose group (250 mg/kg body weight/day) one litter contained four fetuses with an accessory blood vessel associated with the superior pole of the kidney and a second litter contained one small foetus with cranial abnormalities and immature gonads. The isolated nature of these observations did not suggest any association with treatment.

Skeletal Examination

Compared with the concurrent controls and the low dose group (40 mg/kg body weight/day), there appeared to be indications of a reduction in foetal ossification in the high dose group (250 mg/kg body weight/day) and, to a lesser extent, in the intermediate dose group (100 mg/kg body weight/day). Parameters affected included a number of cranial bones, viz. supraoccipital, interparietal, squamosal, jugal. maxilla(e) and hyoid, the caudal vertebral arches and humerus(i). Other ossificationparameters were similar in all groups.

Morphological Examination

A number of foetuses in all groups showed minor morphological changes, but there were no indications of any consistent treatment or dose related associations or trends.

Dams treated with 250 mg/kg bw/d showed maternal toxicity comprising clinical signs, reduced body weight gain and food consumption during the treatment period.Based on the results of this study, DOPA-Glycinate (20% a.i.) is considered not to be teratogenic when orally administered to Wistar rats at levels up to and including 40 mg/kg bw/d based on an increase in incidence of unilateral hydroureter compared with the concurrent controls in conjuction with interparietal and supraoccipital delayed ossification in the middle and high dose group. There was also a slight increase in unilateral renal pelvic cavitation with treatment. There were no increases recorded in the incidences of bilateral hydroureter or bilateral renal pelvic cavitation.

The maternal no observed adverse effect level (NOAEL) was 100 mg/kg bw/d, corresponding to 20 mg a.i./kg bw/d. The NOAEL for survival, growth and development in utero was 40 mg/kg bw/d, corresponding to 8 mg a.i./kg bw/d.

Furthermore, a two generation study with the registration substance is available. The registration substance (20% a.i., as manufactured) was administered to groups of male and female Wistar rats at 0 (control), 10, 30 or 100 mg/kg bw/day in water, corresponding to 2, 6 and 20 mg a.i./kg bw/day, by gavage during the pre-mating and mating period and to females also during gestation and lactation. The two generation reproduction study was carried out in compliance with OECD guideline 416 (1983) and also with the recent version OECD 416 (2001), except that the examination of oestrus cycle and sperm parameters were not conducted in the study. Details on parental toxicity and reproduction is given above.

Developmental toxicity was assessed by observing clinical signs, body weights and macroscopic examination of the pups during their lactation period. Clinical signs, body weights and macroscopic examination were unaffected in F1- and F2-pups with maternal treatment up to 100 mg/kg bw/day. 

Developmental NOAEL= 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day. No treatment-related macroscopic pathological effects were observed for any dose tested. 

Conclusion

The NOAEL(teratogenicity, embryotoxicity) from developmental toxicity studies according to OECD 414 was 30 mg a.i./kg bw/day in rabbits and 8 mg a.i./kg bw/day in rats, respectively. A two generation study according to OECD 416 is available as well. The NOAEL for developmental toxictiy from this OECD 416 was observed to be 20 mg a.i./kg bw/day.


Justification for classification or non-classification

In conclusion, the results of the available data on reproductive and developmental toxicity indicate that the registration substance, does not need to be classified for reproductive and developmental toxicity according to EU GHS (Regulation 1272/2008/EC) and GHS-UN and therefore labelling is not necessary.