Adipic acid, compound with hexane-1,6-diamine (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

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 Type:

publication

Title:

A Two-Generation Reproduction Study in Rats Receiving Diets Containing Hexamethylenediamine.

Author:

Short RD et al.

Year:

1991

Bibliographic source:

Fund Appl Toxicol 16: 490-494.

Materials and methods

Principles of method if other than guideline:

Sprague-Dawley rats were treated with the test compound by oral feeding over two generations for about 40 weeks. Litters were examined for size, stillbirths, live births, and gross anomalies. Pubs were selected for F1 parents of the F2 offspring. After 98 days of treatment the F1 parents were mated. F2 pubs were killed on day 21. Gross necropsies were performed on F0 and F1 parents as well as F2 pubs. Histological evaluation of tissue of different organs was conducted.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylenediamine

EC Number:

204-679-6

EC Name:

Hexamethylenediamine

Cas Number:

124-09-4

Molecular formula:

C6H16N2

IUPAC Name:

hexane-1,6-diamine

Details on test material:

Component of the test substance.

- Name of test material (as cited in study report): 1,6-hexanediamine, 78% aqueous solution
No further data.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI)
- Age at study initiation: (P) ca. 8 weeks; (F1) 3 weeks
- Housing: individually in wire mesh cages or plastic cages with wood chip bedding; except for the 20-day cohabitation (mating)
- Diet (ad libitum): control or experimented diet prepared with ground Purina Certified Rodent Chow No. 5002 (Ralston Purina Co., St. Louis, MO)
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
All rats were housed in environmentally controlled rooms; no further details
- Photoperiod (hrs dark / hrs light): 12/12

No further data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

water

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets for the control, low, and mid dose groups were prepared weekly, while the diet for the high dose group was prepared every 4 days in order to maintain adequate levels of test material.
- Mixing appropriate amounts with (Type of food): ground Purina Certified Rodent Chow No. 5002

A weighed amount of test material was diluted with ethanol and mixed with ground Purina Certified Rodent Chow to form a premix. This premix was further diluted with additional feed to obtain diets formulated to provide 0, 50, 150, and 500 mg/kg/day of hexamethylenediamine. Dietary concentrations were based on the most recent weekly body weights except during the 20-day mating period when concentrations were based on the most recent female values obtained prior to mating.
Diets were analyzed for homogeneity, stability, and concentration of test material.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 20 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diets were analyzed for homogeneity, stability, and to the concentration of test material. A dansylated derivative of hexamethylenediamine was prepared and quantified at 254 nm using a UV detector.

Duration of treatment / exposure:

Exposure period: over two generations
Duration of test: 40 weeks

Frequency of treatment:

continuously in the diet

Details on study schedule:

After a minimum of 56 days of treatment, the F0 rats were mated to produce the F1 offspring.
After a minimum of 98 days of treatment the F1 parents were mated to produce the F2 offspring.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

26 males and 26 females per group

Control animals:

yes, plain diet

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: No data

Oestrous cyclicity (parental animals):

no

Sperm parameters (parental animals):

no

Litter observations:

Pregnant F0 females were allowed to give birth to F1 pups and the day all pups were delivered was designated Day 0 of lactation.
Litters were examined for size, stillbirths, live births, and gross anomalies. Litter size was reduced to a total of 8 pups of equal sex, when possible, on Day 4 of lactation. Pups were housed with their mothers and weighed at intervals for 3 weeks after birth. Afterward, 26 pups of each sex from group were selected to become F1 parents of the F2 offspring.

Postmortem examinations (parental animals):

Gross necropsies were performed on F0 and F1 parents as well as F2 pups. The following tissues were taken from F0 and F1 rats for histopathological evaluation: kidneys, liver, lung, ovaries, prostate, seminal vesicles, spleen, testes with epididymes, uterus, and vagina. Tissues were preserved in neutral buffered formalin and stained with hematoxylin and eosin. No further data.

Postmortem examinations (offspring):

The F2 pups were sacrificed on day 21 of lactation and subjected to gross necropsy. No further data.

Statistics:

Several different statistical methods were used to compare measurements made on test animals to the corresponding values determined for controls. The methods and the measurements to which they applied were analysis of variance and Dunnett's test for body weight and litter size, Chi square test with Yates' correction or Fisher's exact probability test for fertility indices; and Mann-Whitney U test for pup survival. The level of significance was selected as p < 0.05.

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

body weight and weight gain

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: reduced litter size

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

No treatment-related mortality was observed in any of the groups.

The ability of rats to successfully mate and produce litters was not adversely affected by daily doses of up to 500 mg/kg.
The weight of male F0 and F1 parent animals was significantly reduced by  about 10% at 500 mg/kg at the end of the treatment period. The body  weight of the females was not altered at that time but the weight gain  was reduced by about 10% during gestation (no further data).

The litter size at birth was significantly reduced in the F1 generation (13.8 vs 11.7) and not significantly reduced in the F2 generation (13.0 vs. 11.0)  at 500 mg/kg. Pup weight was normal at birth but was significantly lower at day 21 in male F1 pups and female F2 pups at 500 mg/kg. There was no effect on their survival and they appeared normal during lactation. 
The treatment with up to 150 mg/kg did not adversely affect reproduction or fertility. No differences between control and treated rats in either generation with regard to clinical observations, copulation, gestation length, nesting behaviour, appearance of pups. No treatment related effects on testes weights, no microscopic and macroscopic effects on tissues.
No data are given on malformations.

Table 7.8.1/2: F0 Males: Group Mean Body Weight, Standard Deviation and Survival.

	Hexamethylene Diamine (mg/kg/day)
Week of Study	0 (Control)			50			150			500
	Mean Body Weight (g)	± S.D.	Survival	Mean Body Weight (g)	± S.D.	Survival	Mean Body Weight (g)	± S.D.	Survival	Mean Body Weight (g)	± S.D.	Survival
0	269	11.4	26/26	274	10.1	26/26	272	11.6	26/26	267	10.0	26/26
1	317	15.4	26/26	320	13.8	26/26	320	15.7	26/26	308	14.4	26/26
2	347	17.3	26/26	351	17.3	26/26	352	19.3	26/26	332	15.8	26/26
3	371	19.2	26/26	376	19.8	26/26	381	22.3	26/26	353	16.9	26/26
4	392	21.5	26/26	398	22.1	26/26	401	25.8	26/26	367	18.7	26/26
5	415	25.1	26/26	422	23.2	26/26	424	27.9	26/26	389	20.4	26/26
6	432	26.6	26/26	438	26.8	26/26	438	33.9	26/26	404	20.9	26/26
7	446	30.1	26/26	451	31.6	25/26	454	33.0	26/26	413	21.9	26/26
8	458	34.8	26/26	464	28.7	25/26	467	38.2	26/26	421**	23.6	26/26
9	452	34.1	26/26	465	30.8	25/26	468	37.5	26/26	425	22.7	26/26
10	470	34.1	26/26	476	30.8	25/26	474	39.3	26/26	429	23.1	26/26
11	482	35.0	26/26	486	30.5	25/26	486	42.0	26/26	438	25.5	26/26
12	488	35.0	26/26	487	47.6	25/26	492	44.8	26/26	439	26.8	26/26
13	501	36.7	26/26	510	35.2	25/26	505	45.8	25/26	446	27.5	26/26
14	509	38.8	26/26	519	36.8	25/26	515	44.7	25/26	450	28.1	26/26
15	513	39.3	26/26	523	36.9	25/26	521	46.4	25/26	450**	26.4	26/26

** Significantly different from the control group, p < 0.01

S.D. : Standard Deviation

Table 7.8.1/3: F0 Females: Group Mean Body Weight, Standard Deviations and Survival

	Hexamethylene Diamine (mg/kg/day)
Week of Study	0 (Control)			50			150			500
	Mean Body Weight (g)	± S.D.	Survival	Mean Body Weight (g)	± S.D.	Survival	Mean Body Weight (g)	± S.D.	Survival	Mean Body Weight (g)	± S.D.	Survival
0	193	10.1	26/26	194	11.2	26/26	199	8.9	26/26	1199	10.7	26/26
1	216	12.1	26/26	216	13.5	26/26	222	10.0	26/26	220	12.3	26/26
2	226	18.1	26/26	229	15.2	26/26	236	13.7	26/26	229	12.9	26/26
3	238	11.9	26/26	239	18.1	26/26	247	13.0	26/26	239	12.9	26/26
4	247	14.1	26/26	248	17.5	26/26	258	21.7	26/26	249	15.8	26/26
5	256	14.2	26/26	260	20.5	26/26	265	14.7	26/26	259	13.9	26/26
6	265	16.2	26/26	265	19.9	26/26	274	13.0	26/26	263	15.0	26/26
7	272	15.9	26/26	274	21.4	26/26	263	15.7	26/26	268	14.6	26/26
8	274	15.2	26/26	276	22.4	26/26	286*	16.6	26/26	270	16.5	26/26
9	289	15.5	26/26	291	20.0	26/26	298	17.5	26/26	282	14.1	26/26
10	310	17.9	26/26	315	23.7	26/26	320	21.8	26/26	299	14.9	26/26
11	351	31.5	26/26	358	36.3	26/26	356	36.7	26/26	343	26.6	26/26
12	325	23.7	26/26	327	33.1	26/26	327	21.9	26/26	309	19.1	26/26
13	34	29.3	26/26	352	28.5	26/26	347	30.3	26/26	325	22.7	26/26
14	334	22.5	26/26	342	25.1	26/26	343	22.6	26/26	343	26.9	26/26
15	311	22.5	26/26	315	25.2	26/26	317	16.9	26/26	314	17.0	26/26
16	310	22.2	26/26	314	19.4	26/26	317	17.7	26/26	312	18.6	26/26
17	314	17.4	26/26	321	21.5	26/26	321	18.7	26/26	312	18.7	26/26
18	315	18.1	26/26	321	19.9	26/26	325	19.5	26/26	310	20.1	26/26

* Significantly different from the control group, p<0.05

S.D. : Standard Deviation

Applicant's summary and conclusion

Executive summary:

In a two generation study 26 males and 26 females SD- rats/group received Hexamethylenediamine (HMD) orally in the diet at dosage levels of 0, 50, 150 or 500 mg/kg/day for 56 days prior to mating, then throughout the study in accordance with Series 83 -4 of the Environmental protection Agency Pesticide Assessment Guidelines, Subdivision F., Hazard Evaluation: Human and Domestic Animals, issued November, 1982 (similar to OECD 416) and in accordance with GLP.

The parental rats and pups were observed twice each day for signs of overt toxicity, changes in general appearance and behavior, and mortality. Individual body weights were recorded weekly for the adult rats; In addition, females were weighed on gestation days 0, 6, 15 and 20 and lacation days 0, 4, 7, 14 and 21. Parental food consumption was measured weekly for individual parental rats except during mating. Specific reproductive observations included tabulation of male and female fertility indices, and the length of cohabitation and gestation were recorded.Gross necropsies were performed on F0 and F1 parents as well as F2 pups. The following tissues were taken from F0 and F1 rats for histopathological evaluation: kidneys, liver, lung, ovaries, prostate, seminal vesicles, spleen, testes with epididymis, uterus, and vagina.

The results of this study were:

- Dietary analysis indicated that greater than 90% of the target concentration of hexamethylenediamine were fed to rats in all groups. The actual doses consumed, however, averaged between 123 and 132% of the target doses in these groups.
- No treatment-related mortality was observed in any of the groups. Some deaths occurred, however, they were singular events in specific groups and there was no pattern indicative of a dose-response relationship. Mortality ratios for the F0 males were 0/26, 1/26, 1/26, and 0/26 for the 0, 50, 150, and 500 mg/kg groups, respectively. Mortality ratios for the F0 females were 0/26, 0/26, 0/26, and 0/26 for the 0, 50, 150, and 500 mg/kg groups, respectively. Mortality ratios for the F1 males were 0/26, 0/26, 0/26, and 0/26 for the 0, 50, 150, and 500 mg/kg groups, respectively. Mortality ratios for the F1 females were 1/26, 1/26, 0/26, and 0/26 for the 0, 50, 150, and 500 mg/kg groups, respectively.
- Body weights of male F0 and F1 rats in the 500 mg/kg group were reduced by about 10%, relative to control values, at the end of study weeks 15 and38. The body weights of females, in contrast, were comparable to control values at these intervals. During gestation, the female weight gain was reduced by about 10% in the high-dose group. Decreased body weight is correlated with a decreased food consumption. Therefore, this effect was likely due to the unpalatability of HMD.

-Fertility was not adversely affected by the dietary administration of hexamethylenediamine over 2 generations (See Table ). The F0 and the F1 litter size in the 500 mg/kg group was significantly reduced without an increase in the number of dead pups. There was no biological meaningful or statistically significant differences in the number of viable and dead pups on lactation day 1, as compared to control for either generation in the mid and low-dose treatment groups. Pup survival was not significantly reduced in any of the treated groups. At birth, pup body weights were not adversely affected by treatment, but during lactation, reduced weights were apparent in pups of each sex from the high dose group

-No meaningful differences were noted between the control and treated rats of either generation with regard to antemortem observations, copulatory interval, gestation length, nesting and nursing behavior, and appearance of the pups. No treatment related effects were noted on testes weights and no effects were noted by macroscopic or microscopic examination of tissues evaluated.