Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine

Toxicity to reproduction

Administrative data

Endpoint:

two-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:

supporting study

Study period:

From 28 OCT 1983 to 7 AUG 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is conducted according to EPA guidelines (F, 83-4), similar to OECD 416. Study well conducted, no deviation from the protocol. Analytical certificate is available

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Reference 2

Reference Type:

publication

Title:

A two-generation reproduction study in rats receiving diets containing Hexamethylenediamine

Author:

Short RD, Johannsen FR and Schardein JL

Year:

1991

Bibliographic source:

Fundamental and Applied Toxicology, 16:490-494

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

No additional data

GLP compliance:

yes

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:

- Name of test material (as cited in study report): Hexamethylenediamine (1,6-hexanediamine; HMD)
- Substance type: no data
- Physical state: clear liquid
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: at room temperature

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 COBS rats from the Charles River Breeding Laboratories ( Potage, Michigan)
- Age at study initiation: (P) x 8 wks, (F1): 14 wks
- Weight at study initiation: (P) Males: 250-289 g ; Females: 175-214 g, (F1) Males: 114-135 g; Females: 130 - 172 g
- Fasting period before study: no data
- Housing: in suspended wire-mesh cages/plastic bedding in an environmentally controlled room
- Diet: ad libitum, Ground Purina Certified Rodent Chow® 5002 (Ralston Purina Co., St. Louis, MO, USA) during acclimatation period and throughout study
- Water: ad libitum
- Acclimation period: 17 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): a range of 18.3°C to 23.9°C with a mean temperature of 22.1 ± 0.39°C
- Humidity (%): between a range of 24% through 82% with a mean relative humidity of 46.8 ± 13.19%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Administration / exposure

Route of administration:

oral: feed

Vehicle:

ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Not applicable

DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets for the control, low and mid doses were prepared weekly, while the diet for the high dose was prepared every 4 days in order to maintain adequate levels of test material.
- Mixing appropriate amounts with (Type of food): the test article/ethanol solution was premixed with ground Purina Certified Rodent Chow® #5002 for 15 minutes. The resulting premix was added to a twin shell blender with additional ground Purina Rodent Chow® #5002.
- Storage temperature of food: at room temperature


VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): not applicable
- Purity:no data
- Volume of the vehicle: nearest 5 mL

Details on mating procedure:

- M/F ratio per cage: 1M/1F
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to study initiation, test samples of treated diets were prepared in order to assess the homogeneity of the mixed diets and stability of the test article in the diet to be used. Diets were analyzed for homogeneity, stability, and concentration of the test material. Stability analysis showed that the lower concentrations (0.640 and 2.74 g/kg feed) had 10-day stabilities of 95 to 103%; however, the higher concentration (16 g/kg feed) only showed a 10-day stability of 72%. The higher concentration was adequately stable for 4 days (<14% loss).

Duration of treatment / exposure:

- Premating exposure period (males and females): 56 days for F0 and 98 days for F1
- mating period: each male was cohabitated with a female from the same treatment groups for up to 20 days, (both generation), after the matin period, males were individually housed and continued on treatment until the completion of parturition, when fertility was evaluated
- Gestation period: 0-19 days (both generation)
- Lactation period: 3 weeks (F0 and F1 parents)

Frequency of treatment:

Daily, 7 days/week (continuously in feed)

Details on study schedule:

- F1 parental animals mated after minimum of 98 days of treatment.
- Selection of parents from F1 generation when pups were 21 days of lactation.
- Age at mating of the mated animals in the study: no data

Doses / concentrations

Remarks:

Doses / Concentrations:
Doses: 0, 50, 150 and 500 mg/kg b.w./day
Basis:
nominal in diet

No. of animals per sex per dose:

26 rats/sex/dose

Control animals:

yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (twice)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the parental rats were observed twice each day for signs of overt toxicity, changes in general apperance and behavior and mortality. Detailed observations were recorded weekly for the parental rats

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly. In addition, females were weighed on gestation days 0, 6, 15 and 20 and lactation days 0, 4, 7, 14 and 21.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Parental food consumption was measured weekly for individual parental rats except during mating when the males and females were cohabitated.
- 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: Yes

Oestrous cyclicity (parental animals):

Yes

Sperm parameters (parental animals):

Parameters examined in all/P/F1 male parental generations: yes
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed postpartum (day 4 of lactation): yes, if possible, the litter was reduced to an aqual number of males and females (4M/4F).
- If yes, maximum of 8 pups as nearly as possible; excess pups were killed and discarded.


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


GROSS EXAMINATION OF DEAD PUPS:
yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: they were sacrified and necropsied, after mating period and after have continued on treatment until completion of parturition
- Maternal animals: F0 females sacrified and necropsied after selection of F1 parents, F1 females sacrified and necropsied after necropsy of F2 pups.


GROSS NECROPSY: Yes, performed for all F0 and F1 parents and all animals which died on study. Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGTHS:
The following tissues were weighed: Testis , kidney, ovary, seminal vesicle and testis with epididymis
The following tissues were taken fromF0 and F1 for histopathological evaluation: Kidney, liver, lung, ovary, prostate, seminal vesicle, spleen, testis with epididymis, uterus and vagina

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrified on days 21 of lactation.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsyconsisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS:
Organ weights: not perfomed
Histopathology: only two pups with macroscopic findings (see details in results section)

Statistics:

All statistical analyses compared the treatment groups with the control group, with a level of insignifiance at p < 0.05 and p 0.01. All mean are accompained by standard deviations.
The parental body weights by sex were analysed by one-way analysis of variance, Barlett's test for homogeneity of variances, and the appropriate t-test as described by Steel and Torrie. Significant differences were determined using Dunnett's multiple comparison tables.
The number of live born pups per litter and body weights of pups were analysed by one-way analysis of variance, Barlett's test for homogeneity of variances, and the appropriate t-test as described by Steel and Torrie. Significant differences were determined using Dunnett's multiple comparison tables.Male and female fertility indices were compared using the Chi-square tset criterion with Yates' correction for 2 × 2 contingency tables and/or Fisher's exact probability test as described by Siegle to judge levels of significant diferences. The proportion of live pups at birth per total number born and the survival indices at lactation days 0, 4, 7, 14 and 21 were compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significant differences.

Reproductive indices:

Copulation index (%)
Fertility index (male and female; %)
Pregnancy rate (%)

Offspring viability indices:

Mean litter size
mean % born alive
0-4day viability (%)
weaning viability index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

in high dose group: transient body weight reduction in male parental animals of F0 and male and femaleparental animals of F1 generation (effects were not accounted for being adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

in high dose group: transient body weight reduction in male parental animals of F0 and male and femaleparental animals of F1 generation (effects were not accounted for being adverse)

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: in high dose group: in male and female F0 and F1 animals reduced food consumption due to unpalatablility of HMD containing diet

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
1- F0 parents:
- Mortality: two males died, one in the low-dose group (week 8) and one in the mid-dose group (week 14), no mortality was observed in the control and high dose groups. The cause of death was not determined either of these animals. In females, the rate of survivors was 100% in control and all treated animals.
- Clinical signs: in both sexes, no clinical signs - related to treatment were observed. In males, randomly occurring antemortem observations included eye irritation, soft stool, malaligned, broken, or missing upper incisors and long teeth that required trimming. In females, hair loss, malaligned or long teeth were observed in every female group, but occurred in no pattern of frequency that suggested a treatment-related effect.
2- F1 parents:
- Mortality: Two females died, one in the control group (week 35) and one in the low dose group (week 32), no mortality was observed in the mid and high dose groups. The cause of death was not determined either of these animals. In males, the rate of survivors was 100% in control and all treated animals.
- Clinical signs: in both sexes, no clinical signs related to treatment were observed.
In males, dental aberrations, eye irritation and incidental hair loss were observed in all groups.
In females, hair loss was the most frequently noted abnormality in all study groups. Dental abnormalities were seen in the mid and high dose groups (short and misaligned incisors, long teeth). Ocular aberrations were noted in all groups (larger than normal eyes, eyes half closed...).
However, none of these observations of either the F1 males or females were deemed a consequence of treatment due to low incidence and/or comparable control groups findings.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
1- F0 parents:
- Body weight: In the male high-dose group, inhibition of weekly body weight gains occurred throughout the entire generation. A statistically significant decrease on body weight was observed in males treated at high dose. No changes were observed in males treated at low and mid doses.
in females, no significant differences were found between the values of the treated groups and that of the control (at week 18, all gestation period and lactation period)
- Food consumption:
Weekly male food consumption in the high-dose group was decreased in comparison with the control group values during all F0 study weeks.
For the low and mid dose groups, food consumption was comparable to control groups.
In females, for most of the study weeks, weekly female food consumption in the high-dose group was lower than the control group values. In general, similar findings were observed in both male and female food consumption calculated as g/kg/day
2- F1 parents:
- Body weight: In the male high-dose group, inhibition of weekly body weight gains occurred throughout the entire generation. A statistically significant decrease on body weight was observed in males treated at high dose. No changes were observed in males treated at low and mid doses.
Similar findings observed in males were noted for the F1 females.
- Food consumption: See food consumption for F0 parents cited above (the same results were found)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no apparent adverse effects related to the administration of Hexamethylene Diamine observed in female fertility indices at any treatment level (F0 and F1)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no apparent adverse effects related to the administration of Hexamethylene Diamine observed in male fertility indices at any treatment level (F0 and F1)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The lenght of gestation and copulatory interval were not affected by administration of hexamethylene diamine at any of the levels tested (F0 and F1)

ORGAN WEIGHTS (PARENTAL ANIMALS)
The only organs weighed were testes of males on which spermatogenesis evaluation was conducted because of failure to sire a litter. There were no test substance related differences in the mean absolute or relative testicular weights between control and experimental groups (F0 and F1).

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test substance related macroscopic changes were observed in male or female rats which died during the course of this study or which were sacrified after full term exposure to 0, 50, 150 or 500 mg/kg/day of hexamethylene diamine in the diet (F0 and F1)

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no test substance related microscopic changes observed in male or female rats which died during the course of this study or which were sacrified after full term exposure to 0, 50, 150 or 500 mg/kg/day of hexamethylene diamine in the diet (F0 and F1)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Slightly decreased pup weights observed at high dose. It could be attributed to the unpalatability of HMD thus inducing decrease in food consumption by the F0 and F1 parents. Therefore this effect was not accounted for being adverse.

Remarks on result:

other: Generation: Developmental (migrated information)

3

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: reproduction and fertility (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Significant but only slight decrease in litter size in the high dose group which is considered to be still in the range of the control group (even without historical control data).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slight reduction of body weight of F1 and F2 pups from high dose groups compared to control pups at PND 21 (statistically significant only for F1 male pups and F2 female pups)

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
- A statistically significant reduction in viable litter size was observed in the high-dose group, no corresponding increase in the number of dead pups was noted. There were no biologically meaning ful or statistically significant differences in the number of viable and dead pups on lactation day 0 in the low and mid dose groups in comparison with the control group (F0 and F1)
Details:
The mean number live F1 pups at birth is 14; 15.2; 13.1 and 12 respectively for control, low, mid and high dose groups. The reduction of F1 litter size at birth is not statistically significant. Moreover, this effect observed didn’t correspond to an increase in the number of dead pups or to an impact on reproductive parameters.
The mean number live F2 pups at birth is 13; 13.5; 13.1 and 11 respectively for control, low, mid and high dose groups. The reduction of F2 litter size at birth is statistically significant. But this reduction is slight and no historical controls are available in the study.
- Pup survival to weaning indices of the treated and control groups were not statistically different.

CLINICAL SIGNS (OFFSPRING)
The general appearance and behavior of pups in the treated groups were not different from those in the control group.

BODY WEIGHT (OFFSPRING)
- Pup weight at birth in all the treated groups was comparable to the control group value (for both two generation study).
- Pup weight (males and females) of the high-dose group on lactation day 14 was significantly lower than the control group value. Similarly, the male pup weight of the high-dose group on lactation day 21 was also significantly lower than the control group value. Lactation day 21 female body weights of the high-dose group were lower in comparison with the control value, but no statistically significant difference was noted. Pup body weights on lactation days 4 and 7 in the high dose group and on lactation day 4, 7, 14 and 21 in the low and mid dose groups were comparable to the control group values.
For F1 male pups the mean weight at day 21 of lactation is 53 and 49 grams respectively for the control and the high dose groups. This reduction is statistically significant.
For F1 female pups the mean weight at day 21 of lactation is 50.9 and 47.6 grams respectively for the control group and the high dose group. This reduction is not statistically significant.

For F2 male pups the mean weight at day 21 of lactation is 53.6 and 51 grams respectively for the control and the high dose groups. This reduction is not statistically significant.
For F2 female pups the mean weight at day 21 of lactation is 51.5 and 49 grams respectively for the control group and the high dose group. This reduction is statistically significant.

SEXUAL MATURATION (OFFSPRING)
no data

ORGAN WEIGHTS (OFFSPRING)
not perfomed

GROSS PATHOLOGY (OFFSPRING)
No treatment-related effects were observed . Macroscopic liver findings were described in two F1 pups (1 male and 1 female), therefore a histopathology was recorded (see results cited below)

HISTOPATHOLOGY (OFFSPRING):
No treatment related effects were observed.
Hydronephrosis and or distended ureters were observed in pups (day 4 and 21 of lactation), however, these findings were considered as not treatment related, it were also observed in control groups.
Microscopically, the macroscopic liver findings observed in two F1 pups (cited above) consisted of moderate fibrosis in the hepatic capsule of the female and extensive (generalized) extramedullary hematopoiesis in both pups. However, hematopoiesis in the liver is normal for rat pups at this age.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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

Conclusions:

Under the test conditions, there were no adverse effects on reproduction and fertility induced by HMD. Therefore,
The NOEL (parental) = 500 mg/kg bw/day (Overall effects observed (i.e. transient body weight reduction in male parental animals of F0 and male and female parental animals of F1 generation, reduced food consumption due to unpalatablility of HMD containing diet in male and female F0 and F1 animals) were not accounted for being adverse as they were of transient nature and only about 10%).
The NOAEL (Developmental) = 500 mg/kg bw/day, (Slightly decreased pup weights observed at high dose, which could be attributed to the unpalatability of HMD thus inducing decrease in food consumption by the F0 and F1 parents. Therefore this effect was not accounted for being adverse. ).
The NOAEL (Fertility) = 500 mg/kg bw/day, (Significant but only slight decrease in litter size in the high dose group which is considered to be still in the range of the control group and there were no other effects observed (e.g. no increase in number of dead pups or any other impacts on reproductive paramteres).
In addition, these results indicate that HMD does not have significant systemic target organ toxicity.

Executive summary:

In a two generation study (Schardein, 1985), 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 lactation 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 and 38. 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. 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 were 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.

In conclusion:

Under the test conditions, there were no adverse effects on reproduction and fertility, therefore:

The NOAEL (Parental) = 500 mg/kg bw/day (with NOEL = 150 mg/kg bw/day, even if the transiently decreased body weights of F0 males and F1 male and female animals were likely due to the decrease of food consumption as the worst case)

The NOAEL (Developmental) = 500 mg/kg bw/day (with NOEL = 150 mg/kg bw/day, since there were slightly decreased pup weights observed at high dose in comparison to the control, which could be attributed to the unpalatability of HMD thus inducing decrease in food consumption by the F0 and F1 parents).

The NOAEL (Fertility) = 500 mg/kg bw/day (with NOEL = 150 mg/kg bw/day, based on significant but slight decrease in litter size in the high dose group which is considered to be in the range of the control group (even in the absence of historical control data) and without any other effects on reproduction).