Hexahydro-2H-azepin-2-one, sodium salt

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Reference 2

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

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:

yes

Remarks:

(Hazleton Laboratories America, Inc.)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

ε-caprolactam

EC Number:

203-313-2

EC Name:

ε-caprolactam

Cas Number:

105-60-2

Molecular formula:

C6H11NO

IUPAC Name:

azepan-2-one

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 or test system 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 / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

5 000 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

10 000 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day

Remarks:

calculated based on a conversion factor of 10 (Derelanko, M.J., The Toxicologist's Pocket Handbook, 2nd Ed., p.29, 2008).

Doses / concentrations 5

Dose / conc.:

500 mg/kg bw/day

Remarks:

calculated based on a conversion factor of 10 (Derelanko, M.J., The Toxicologist's Pocket Handbook, 2nd Ed., p.29, 2008).

Doses / concentrations 6

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

calculated based on a conversion factor of 10 (Derelanko, M.J., The Toxicologist's Pocket Handbook, 2nd Ed., p.29, 2008).

Doses / concentrations 7

Dose / conc.:

79 mg/kg bw/day (actual dose received)

Remarks:

females

Doses / concentrations 8

Dose / conc.:

355 mg/kg bw/day (actual dose received)

Remarks:

females

Doses / concentrations 9

Dose / conc.:

726 mg/kg bw/day (actual dose received)

Remarks:

females

Doses / concentrations 10

Dose / conc.:

86 mg/kg bw/day (actual dose received)

Remarks:

males

Doses / concentrations 11

Dose / conc.:

422 mg/kg bw/day (actual dose received)

Remarks:

males

Doses / concentrations 12

Dose / conc.:

878 mg/kg bw/day (actual dose received)

Remarks:

males

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 generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted within the parental generations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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 weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.
No effects on body weight and food consumption were noted in the rats treated at 1000 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

The pregnancy and fertility indices were comparable between the treated and control groups for each filial generation, and no dose-related trends were apparent.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical observations were noted in any of the three filial generations.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

no treatment related effects were observed

Other effects:

no effects observed

Description (incidence and severity):

No treatment-related effects were noted in evaluation of the percentage of male pups.

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.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F2)

Critical effects observed:

not specified

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 ratios^a, 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