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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles of GLP, acceptable with restrictions.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981
Reference Type:
publication
Title:
Three-Generation Reproduction Study with Caprolactam in Rats.
Author:
Serota, D.G. et al.
Year:
1988
Bibliographic source:
J. Appl. Toxicol. 8, 285-293

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Additional third generation, missing assay parameters: lenght of estrus cycle, vaginal opening, preputial separation, sperm parameters, anogenital distance
Principles of method if other than guideline:
The effects of Caprolactam on reproduction were studied in a three-generation reproduction study in albino rats. The chemical was administered in the diet at levels of 0, 1000, 5000, and 10000 ppm (0, 100, 500 and 1000 mg/kg bw/d, respectively). Criteria used to evaluate for compound effect in the parental animals were mortality, clinical signs, body weight, food consumption, reproduction indices, and gross and microscopic pathology. Criteria evaluated in the offspring were gross appearance, survival, body weight, male pup percentages, gross pathology and kidney weight data.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Caprolactam
- 3 lots of caprolactam were used. In order to achieve a homogeneous blend, equal amounts of each lot were ground until homogeneous and used to prepare each batch of test diet.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts
- Weight at study initiation: (P) Males: 88-133 g, Females: 67-104 g


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-29
- Humidity (%): 31-68
- Photoperiod (hrs dark / hrs light): 12/12
- Housing in non-breeding period: individually in wire-mesh cages
- Housing in breeding period: one male and two females per breeding cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The compound and feed were mixed in a Hobart mixer which at small amounts gave a better blend. Fresh diets were prepared and presented weekly.
- fresh diest were prepared weekly and reserve samples were retained from each mixed batch and sent to the sponsor for analysis.
Details on mating procedure:
Mating phase of first (P1), second (P2) and third (P3) generation:
10 males and 20 females were mated / group.
- M/F ratio per cage: 1/2
- Length of cohabitation: 2 weeks
- each parental generation was subjected to two breeding phases. The second breeding occurred 7-13 days after the sacrifice of the first litter of
offspring.



Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Treatment phase:
10 weeks-premating exposure period (males and females of P1).
Following mating the treatement phase was 10 weeks.
Dosing of test substance continuously in the diet throughout three successive generations.

Frequency of treatment:
daily
Details on study schedule:
- parental animals were 10 males and 20 females per dose group in each generation. They were treated 10 weeks before mating.

- F1-generations:
F1a
(1) gross necropsy on approx. 1/3 of the pups
(2) remaining pups were sacrificed without necropsy
F1b
(1) 10males and 20 females / group were selected for P2 generation
(2) gross necropsy on approx. 1/3 of the pups
(3) remaining pups were sacrificed without necropsy

- F2-generations:
F2a
(1) gross necropsy on approx. 1/2 of the pups
(2) remaining pups were sacrificed without necropsy
F2b
(1) 10males and 20 females / group were selected for P3 generation
(2) gross necropsy on approx. 1/3 of the pups
(3) remaining pups were sacrificed without necropsy

- F3-generations:
F3a
(1) gross necropsy on approx. 1/3 of the pups
(2) remaining pups were sacrificed without necropsy
F3b
(1) gross necropsy on approx. 1/2 of the pups
(2) remaining pups were sacrificed without necropsy

- Each litter was observed and the number of live and dead pups (by sex), individual body weights and any evidence of abnormality were recorded on days 1, 7 and 21 of lactation . At Day 7, litters were culled by random card draw to eight pups (equal number per sex where possible).
- The second (P2) and third (P3) parenteral animals were selected by random card draw from each group of F1b and F2b litters for assignment to the same treatment group as described above.


Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000, 5000 and 10000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
100, 500 and 1000 mg/kg bw
Basis:
other: calculated based on a conversion factor of 10 (Derelanko, M.J., The Toxicologist's Pocket Handbook, 2nd Ed., p.29, 2008).
Remarks:
Doses / Concentrations:
79, 355, 726 mg/kg bw/day in females; 86, 422, 878 mg/kg bw/day in males
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 20 females
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: any gross signs of toxicity and pharmacologic effects


BODY WEIGHT: Yes
- Time schedule for examinations: initially and at week 4 and 10


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes for weeks 4 and 10
Postmortem examinations (parental animals):
P1 animals were sacrificed and complete gross necropsies were performed.
The P2 and P3 animals were sacrificed and discarded without necropsy.
All parental animals that died on study were necropsied and gross observations recorded.
Postmortem examinations (offspring):
Gross necropsy was performed of 1/3 (F1a/b, F2b, F3a/b) or 1/2 (F3b) of the offspring.
The kidneys from each necropsied F3b pup were weighed and kidney/body weight ratios were determined.
Statistics:
Parental body weights and food consumption values, and offspring body weights from each generation of the control group, were compared statistically to data of the treated groups of the same sex by Bartlett's test for homogeneity of variance and the one-way classification analyses of variance (ANOVA).
If there were significant results, a multiple pairwise comparison procedure or Scheffe's multiple pairwise comparison procedure was used to compare the group mean values. Reproduction and viability indices were analyzed using the chi-square method.
The percentage of male pups at each interval was analyzed using Wilcoxon's non-parametric comparison of group means. The individual litter was considered as the experimental unit in all calculations.
The F3b kidney weight data for the control group were compared statistically to the data for the caprolactam treated groups of the same sex by Bartlett's test for homogeneity of variance, and then by a one-way classification analysis of variance (ANOVA) if the variance proved to be homogeneous. If the variances were heterogeneous, a log10 transformation was performed, followed by Bartlett's test. If the log10 transformation could not remove the variance heterogeneity, a loge transformation of the original data, followed by Bartlett's test, was performed. If homogeneity could not be achieved, the ANOVA of the non-transformed data was completed. If the ANOVA of homogeneous data was significant, Scheffe's multiple pairwise comparison
procedure was used to compare the group mean values. If the ANOVA of heterogeneous data was significant, Games and Howell's multiple pairwise
comparison procedure was used to compare the group mean values. All analyses were evaluated at 5% probability (one-tailed) level.

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No treatment-related clinical signs were noted within the parental generations. Within the P1 animals, one low- and two high-dose females died during Week 1 of the ten-week growth period. One P2 control male was found dead following the growth period and one P3 male was found dead just prior to the F3b breeding. These deaths were noted randomly by test group, sex and parenteral generation, and were not attributed to the administration of caprolactam.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS, see tables 1 and 2):
Significantly (p<=0.05) lower body weights were noted in both the high-dose P2 and P3 males and females at weeks 0, 4, and 10. In addition, generally lower body weights were noted in the mid-dose group of both sexes of the P2 animals, and the male P3 animals at the same intervals, with the mid-dose P2 males at Week 4 being significant.
A similar effect on food consumption was observed although the response in the males appeared to be more pronounced than in the females. Significantly lower food consumption values were noted in the high-dose P1 females at week 10 (17 %), the mid-dose P2 males at weeks 4 and 10 (10-15 %), the high-dose P2 and P3 males at weeks 4 and 10 (9-18%), and the high-dose P2 and P3 females at week 10 (7-10%).
No effects on body weight and food consumption were noted in the rats treated at 1000 ppm.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS, see tables 3-5):
The pregnancy and fertility indices were comparable between the treated and control groups for each filial generation, and no dose-related trends were apparent.


GROSS PATHOLOGY (PARENTAL ANIMALS): no treatment related effects were observed


HISTOPATHOLOGY (PARENTAL ANIMALS): Compound-related histopathologic findings noted in the high-dose P1 animals consisted of a slight increase in the severity of spontaneous nephropathies.
This nephropathy was characterized by the presence of regenerative tubule epithelium, mononuclear inflammatory cells and occasional dilated tubules
containing proteinaceous casts. In addition, dilated tubules containing granular casts were noted in three of the rats. Kidney sections from rats in the control group as well as the low- and mid-dose treated groups revealed slightly less severe nephropathy and were essentially comparable in appearance between the control and treated rats.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight, spontaneous nephropathies
Remarks on result:
other: P-generation
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Generation: Fertility (migrated information)

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING, see tables 3-5): The live birth, neonatal survival and weaning survival indices were generally comparable between the treated and control groups for each filial generation, and no dose-related trends were noted.

CLINICAL SIGNS (OFFSPRING): No treatment-related clinical observations were noted in any of the three filial generations.

BODY WEIGHT (OFFSPRING, see tables 3-5): Lower mean body weights were observed in male and female pups at the two highest dose levels in all filial generations. The response was dose-related, with significant differences (P ≤ 0.05) noted more frequently in the pups receiving a dose of 10000 ppm.

SEX (OFFSPRING): No treatment-related effects were noted in evaluation of the percentage of male pups.

ORGAN WEIGHTS (OFFSPRING): No significant differences were noted in the mean relative kidney weights (Table 6).

GROSS PATHOLOGY (OFFSPRING): no treatment related effects were observed

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1. Summary of maternal reproduction indeces, offspring survival, and growth from the first generation.  

 

F1a

F1b

Dose [ppm]

0

1000

5000

10000

0

1000

5000

10000

Parental reproduction indeces

#females

20

19

20

18

20

19

20

18

#pregnant females

19

15

19

14

19

17

18

16

Pregnancy rate [%]

95

79

95

77

95

89.5

90

88.9

Male fertility rate [%]

100

100

100

100

100

90

100

100

Offspring indeces

Day 1 #pups/litter

9.8 +/-2.2

8.7 +/-2.5

9.1 +/-2.0

9.2 +/-2.5

10.6 +/-4.2

8.8 +/-3.1

10.3 +/-2.8

9.7 +/-3.3

Day 1 live birth index [%]

98.7

100

100

93.8

99.5

100

99.1

100

Day 7 survival index [%]

100

96.2

100

93.9

99

98.6

98.6

97.9

Day 21 survival index [%]

100

99.2

100

91.1

100

100

99.3

100

% males day 1

44.9

46.9

50.9

55.7

51.8

50

46.5

52.9

Mean offspring body weights

Males day 1

5.91 +/-0.89

5.87 +/-0.71

6.07 +/-0.77

4.86 +/-0.36**

5.49 +/-0.73

5.77 +/-1.05

5.22 +/-0.52

5.25 +/-0.66

females day 1

5.56 +/-0.73

5.61 +/-0.68

5.59 +/-0.75

4.53 +/-1.56

5.4 +/-0.71

5.36 +/-0.58

4.79 +/-0.35**

4.86 +/-0.66

Males day 7

10.77 +/-1.28

11.49 +/-2.22

10.78 +/-1.4

8.76 +/-1.69**

11.02 +/-1.17

11.68 +/-1.96

9.94 +/-1.28

9.03 +/-1.55**

females day 7

10.25 +/-1.40

11.11 +/-2.06

10.31 +/-1.09

8.9 +/-1.06**

10.62 +/-1.23

11.09 +/-1.68

9.28 +/-1.17

8.75 +/-1.45**

Males day 21

29.65 +/-2.66

30.46 +/-4.4

27.65 +/-2.98

22.79 +/-1.28**

31.58 +/-2.46

31.48 +/-4.09

27.97 +/-3.8**

25.03 +/-3.35**

females day 21

28.08 +/-2.3

29.11 +/-3.56

26.62 +/-3.05

22.22 +/-1.69**

29.82 +/-2.68

30.08 +/-3.64

25.79 +/-2.26**

24.63 +/-3.28**

**p≤0.05

Table 2. Summary of maternal reproduction indeces, offspring survival, and growth from the second generation.  

 

F2a

F2b

Dose [ppm]

0

1000

5000

10000

0

1000

5000

10000

Parental reproduction indeces

#females

20

20

20

20

20

20

20

20

#pregnant females

16

19

15

17

15

20

18

17

Pregnancy rate [%]

80

95

75

85

75

100

90

85

Male fertility rate [%]

100

100

100

100

100

100

100

100

Offspring indeces

Day 1 #pups/litter

10 +/-2.7

9.2 +/-3.6

9.5 +/-2.8

8.4 +/-2.2

10.3 +/-2.7

10.7 +/-3.2

8.6 +/-3.4

7.8 +/-3.4

Day 1 live birth index [%]

100

98.2

98.1

100

100

99.6

100

98.2

Day 7 survival index [%]

100

100

99.3

100

99.1

99.2

99.2

91.1

Day 21 survival index [%]

100

99.3

100

99.3

100

100

99.3

93.4

% males day 1

54.7

52.6

50.3

53.4

49.3

52.1

47.9

46.6

Mean offspring body weights

Males day 1

5.54 +/-0.7

5.6 +/-0.65

5.46 +/-0.627

4.99 +/-0.94

5.56 +/-0.49

5.48 +/-0.57

5.5 +/-0.59

5.12 +/-0.7

females day 1

5.35 +/-0.61

5.18 +/-0.4

5.09 +/-0.6

4.67 +/-0.78**

5.32 +/-0.47

5.15 +/-0.41

5.16 +/-0.6

4.74 +/-0.63**

Males day 7

11.48 +/-1.85

10.79 +/-1.16

10.7 +/-1.23

9.33 +/-0.82**

10.83 +/-1.37

10.67 +/-1.65

10.24 +/-1.47

9.29 +/-1.79

females day 7

10.87 +/-1.46

10.24 +/-1.03

10.27 +/-1.18

8.75 +/-0.99**

10.26 +/-1.25

9.84 +/-1.84

9.75 +/-1.41

8.76 +/-1.76

Males day 21

31.95 +/-3.46

31.07 +/-2.94

29.69 +/-2.86

24.36 +/-3.26**

31.89 +/-2.39

32.1 +/-3.2

29.47 +/-2.51**

25.03 +/-4.94**

females day 21

30.14 +/-2.99

29.47 +/-2.02

27.73 +/-2.12

22.97 +/-2.51**

30.46 +/-1.97

29.43 +/-3.28

28.32 +/-2.74

23.49 +/-4.43**

**p≤0.05

Table 3. Summary of maternal reproduction indeces, offspring survival, and growth from the third generation.  

 

F3a

F3b

Dose [ppm]

0

1000

5000

10000

0

1000

5000

10000

Parental reproduction indeces

#females

20

20

20

20

20

20

20

20

#pregnant females

14

15

17

16

17

17

15

15

Pregnancy rate [%]

70

75

85

80

85

85

75

83.3

Male fertility rate [%]

90

100

100

100

100

100

100

100

Offspring indeces

Day 1 #pups/litter

9.4 +/-2.6

9.8 +/-3.6

10.8 +/-2.8

9.5 +/-1.6

9 +/-4.5

9.9 +/-4.3

9.1 +/-3.1

7.7 +/-1.9

Day 1 live birth index [%]

99.5

100

100

100

99.5

100

98

96.2

Day 7 survival index [%]

94.1

90.9

98.2

99.3

96.8

94.8

90.5

99.3

Day 21 survival index [%]

96.5

99.1

96.3

99.3

99.3

93.4

98.1

100

% males day 1

47.1

47.3

45.9

56.7

53.3

52.8

57.8

51.9

Mean offspring body weights

Males day 1

5.9 +/-0.51

5.43 +/-0.45

5.27 +/-0.72**

5.17 +/-0.62**

5.79 +/-1.01

5.47 +/-0.92

5.59 +/-1.01

4.93 +/-0.68

females day 1

5.44 +/-0.35

5.15 +/-0.4

4.91 +/-0.78

4.73 +/-0.57**

5.36 +/-0.71

5.04 +/-0.67

5.35 +/-0.95

4.62 +/-0.42

Males day 7

10.29 +/-1.64

10.49 +/-1.7

9.85 +/-1.78

9.09 +/-1.74

10.85 +/-2.22

10.36 +/-2.95

10. 4 +/-2.31

8.86 +/-1.46

females day 7

9.81 +/-1.5

9.89 +/-1.85

9.63 +/-1.79

8.75 +/-1.79

10.28 +/-1.68

9.77 +/-2.47

9.96 +/-2.35

8.11 +/-1.28**

Males day 21

29.96 +/-3.07

30.28 +/-4.35

27.18 +/-2.65**

23.03 +/-3.82**

31.22 +/-3.63

30.46 +/-5.67

28.3 +/-5.08

23.77 +/-4.32**

females day 21

27.61 +/-2.56

28.01 +/-3.36

26.21 +/-2.44

22.49 +/-3.56**

29.22 +/-2.71

28.75 +/-4.41

27.41 +/-4.42

21.96 +/-3.33**

**p≤0.05

Table 4. Mean terminal body weights, kidney weights and kidney/body weight ratiosa, of selected F3b, pups from a three-generation reproduction study of Caprolactam in rats.

Dose (ppm)

No. examined

Terminal body weight (g)

Kidney weight (g)

Kidney/body weight ratio

Males

0

25

30.80±3.571

0.456±0.1163

1.481±0.3432

1000

31

28.84±40.50

0.448±0.1402

1.545±0.4001

5000

26

27.62**±3.060

0.402±0.1045

1.453±0.3094

10000

25

22.32**±2.174

0.318**±0.0378

1.426±0.1416

Females

0

21

27.95±2.397

0.424±0.1248

1.513±0.3946

1000

20

29.40±4.604

0.419±0.0710

1.428±0.1492

5000

20

27.35±4.030

0.427±0.1126

1.555±0.3029

10000

26

21.31±2.739

0.310**±0.476

1.455±0.1083

aEach value shown is the mean± the standard deviation

** p<0.05 vs control

Applicant's summary and conclusion