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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A subchronic oral toxicity study with detailed histopathological examination of the reporductive organs is available (Klimisch score 2). Moreover a two-generation study with one of the main components of the submission substance is at hand (Klimisch score 1). The overall quality of the database is therfor high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

A subchronic inhalation toxicity study with detailed histopathological examination of the reporductive organs is available (Klimisch score 1). Moreover a mating trials following a subchronic inhalation exposure of rats and mice with one of the main components of the submission substance are at hand (Klimisch score 2). The overall quality of the database is therfor high.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the absence of any adverse effects on reproductive organs or tissues in any of the repeated dose study conducted with the submission studies as well as no impairment of reproductive parameters in the developmental toxicity study there is no further study on reproduction required and therefore available for the submission substance.

As supporting data the results of a two generation study on rats and a one generation study on rats and mice testing one of the main constituents of the submission substance is presented.

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 an EPA Guideline (similar to OECD 416) and in accordance with GLP.

Male and female rats received 0, 50, 150 and 500 mg/kg bw/day in the diet, daily over two generations. No treatment- related mortality was observed in any of the groups, the weight gain of parents (F0 and F1) and pups was slightly reduced in the high dose group. The decreased body weight could be attributed to the unpalatability of HMD inducing decrease in food consumption by the F0 and F1 parents. The litter size was slightly reduced at birth in the high dose group, there was no adverse effect on survival during lactation in any of the treated groups. 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).

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).

In conclusion, there were no adverse effects on reproduction and fertility induced by HMD treatment.

The results of the one generation study supports this finding.

Rats and mice were subjected to a one-generation reproduction study, with 1,6 -hexanediamine Dihydrochloride (HDDC). The test material was administered to rats at dose levels of 0, 16, 50 and 160 mg/m3 by whole-body inhalation for 13 weeks. The exposure concentrations corresponded to 0, 10, 31 and 100 mg/m3 of HMD (one of the main constituents of the submission substance).

In rats no reproductive toxicity was observed. There was no effect on male or female fertility, body weight or body weights gains, gestation length, litter size, neonatal survival, pup weights, sex ratios or pups, or pup morphology in rats exposed to HDDC.

In mice no relevant reproductive toxicity was observed. No changes in body weights or body weights gains were recorded for both sexes compared to control. No effect on male and female fertility were observed. A statistically significant increase in the mean gestation lenght of mice in the 50 and 160 mg/m3 exposure groups was noted but without biological significance in the absence of other reproductive toxicity. HDDC had no effect on litter size, neonatal survival, sex ratio of pups, or pup morphology in mice. Pups in the 160 mg/m3 exposure group had mean weights similar to that of controls at birth and on lactation day 5; however, mean weights for pups in this exposure group were lower than that of controls on lactation days 14 and 21.

Short description of key information:
There were no adverse effects on reproductive organs or tissues in repeated dose toxicity studies (28 day or 90 day study) with the submission substanc. Moreover there is a two-generation study in rats using HMD as test material, which is one of the main constituents of the submission substance (oral in feed, similar to OECD 416). 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).
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).

Justification for selection of Effect on fertility via oral route:
No study was selected as evaluation is based upon all observations taken from all subchronic oral toxicity studies. In the absence of any adverse effects on reproductive organs or tissues in any of the repeated dose study conducted with the submission substance studies as well as no impairment of reproductive parameters in the developmental toxicity study there is no further study on reproduction required and therefor available for the submission substance. Only one two-generation study using a test material which is one of the main components of the submission substance was performed and data are presented here.

Justification for selection of Effect on fertility via inhalation route:
No study was selected as evaluation is based upon observations taken from all subchronic inhalation toxicity studies. In the absence of any adverse effects on reproductive organs or tissues in any of the repeated dose study conducted with the submission substance studies as well as no impairment of reproductive parameters in the developmental toxicity study there is no further study on reproduction required and therefor available for the submission substance. Moreover results of mating trials (following a 13 week inhalation study) with rats and mice, using a test material which is one of the main components of the submission substance , are presented here.

Justification for selection of Effect on fertility via dermal route:
Studies after oral and inhalation exposure are available, indicating no adverse effects on reproductive capacity of test animals.

Effects on developmental toxicity

Description of key information

In a Prenatal Developmental Toxicity Study (rat, oral administration of doses: 0, 50, 100 or 250 mg test material/kg body weight on GD 6 to 15). With regard to the present study the respective maternal LOAEL (effects were early deaths as well respiratory rales in the maternal animals; including effects on reproduction and fertility) in this study is 50 mg test material/kg bw/day.  Based on the lack of any effects concerning the foetal development (i.e. no treatment related effects on foetal ossification variations, external, soft tissue or skeletal malformations) the developmental NOAEL of this study is 250 mg test material/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 JUN 1983 to 29 JUL 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: U.S. Environmental Protection Agency. Seection 83–3: Teratogencity Study. Subdivision F. for Hazard Evaluation: Human and Domestic Animals. issued November, 1982.

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

substance exposure only on GD 6-15 instead until one day prior to the schedule kill (i.e. GD20 in this study), slightly higher temperature (1 °C) as indicated in guideline on one day due to power outage

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Charles River COBS CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: 12 weeks (at the time of mating)
- Weight at study initiation: females: 213-274 g (on GD 0)
- Housing: individually housed in hanging wire-mesh cages
- Diet: Purina Certified Rodent chow #5002 (Ralston Purina Company, ST. Louis, Missouri, USA), ad libitum
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26
- Humidity (%): 51-70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of the test item for each day was weighed into a graduated mixing cylinder. A suffucient quantity of the vehicle, deionized water, was added to yield a total volume of 25 ml of prepared material. The cylinder was shaken by hand and dispensed into a capped container.
The test material was prepared fresh daily at a concentration of 0,2 g/ml to permit the administration of dose levels of 50, 100 and 250 mg test material/kg/day at dosage volumes of 0.25, 0.5 and 1.25 ml/kg, respectively.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1
- Length of cohabitation: until copulatory plug was seen at the daily inspection. The day the vaginal plug was observed was defined as gestation day (GD 0)

Duration of treatment / exposure:

from day 6 to 15 of gestation

Frequency of treatment:

once daily

Duration of test:

animals were sacrificed on day 20 of gestation

Remarks:

Doses / Concentrations:
0, 50, 100 and 250 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
0, 40, 80, and 200 mg/kg bw/day
Basis:
actual ingested
calculated without water (approx. 20 %)

No. of animals per sex per dose:

25 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of range finding study

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 9, 12, 16, 20 (day of scheduled kill)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:abdominal and thoracic cavities were examined for grossly evident morphological changes. If any changes were visual, the respective maternal tissues were preserved in 10 % neutral buffered formalin for histopathological examinations. Uteri from females which appeared nongravid were opened and placed in 10 % ammonium sulfide solution for detection of implantations.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of viable and nonviable fetuses

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No

Statistics:

All statistical analyses compared the treatment groups to the control with the level of significance at p<0.05 and p<0.01. All means were accompanied by standard deviations.
Male to female fetal sex ratios and the proportions of litters with malformations were compared using the Chi-square test criterion with Yates' correction for 2 x 2 contingency tables and/or Fisher's exact probability test as described by Siegel to judge significance of differences.
The proportions of resorbed and dead fetuses and postimplantation losses were compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significance of differences.
Mean numbers of corpora lutea, total implantations, postimplantation loss, live fetuses and mean fetal body weights were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance of differences.

Indices:

Group mean preimplantation loss (%) = (total no. corpora lutea - total no. implantations)/total no. corpora lutea*100
Group mean postimplantation loss (%) = (total no. implantations - total no. viable fetuses)/total no.implantations*100

Historical control data:

Available for maternal and fetal observations at cesarean section and for incidence of malformations, developmental and genetic variations.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: mortality (but no particular cause of early death could be identified at necropsy), respiratory difficulties

Details on maternal toxic effects:
Control group: Survival was 100% during this study.
200 mg/kg/day: 4 animals died between GD 7 and 12, 1 rat was sacrificed in extremis on GD 14
80 mg/kg/day : 5 rats died between GD 7 and 20, 1 rat was sacrificed in extremis on GD 12
40 mg/kg/day: 1 rat died at GD 11
The cause of death could not be determined for any of these animals.

The antemortem observations of the animals that failed to survive to study termination included dried black or red matter around one or both eyes, forelimbs, and nose and/or mouth; moist, black material around the vagina; respiratory rales; nasal discharge; labored breathing; vocalization during breathing; emaciated appearance and moribundity (rats that were sacrificed only).

Necropsy findings:
200 mg/kg/day: One female was observed to have dried, red staining around nose and mouth, hydronephrosis and pitting of the right distended right ureter and calculi in the urinary bladder.
80 mg/kg/day animals that died or were sacrificed in extremis had stomach erosions; distended esophagus, stomach, intestines ureter; hydronephrosis; and mucoid material in the intestines.
40 mg/kg/day: No lesions were exhibited in the rat of this dosage group that died prior to study termination.

Respiratory rales occurred in all treated groups and was considered compound related.

Other findings (e.g. hiar loss, dry matter around eyes, nose, slightly reduced body weight gains during first treatment interval (GD6 to9) etc.) were not considered meaningful, as they were either not dose-dependent, the findings exhibited a high degree of variability or were observed also in the negative control group.

Findings concerning reproduction:
40 and 80 mg/kg/day: slight decrease for mean numbers of corpora lutea and total implantations and increases in postimplantation losses resulted in decreased numbers of viable fetuses in these groups. Effect in low dose group is based on findings in only one out of 25 animals which had 11 late resorptions while all the other animals in the group had no late resorptions. In the mid dose group two out of 25 animals showed either 13 or 11 late resorptions while all other animals showed no increase compared to controls.
200 mg/kg/day: No notable differences were seen in the above mentioned parameters in comparison to the controls.
Since there was no effect at the high dose and no dose-related trend as the 40 mg/kg/day appeared to be marginally more affected than the 80 mg/kg/day
group with respect to the decrease in mean corpura lutea number, total implantations and numbers of viable fetuses, the findings were not considered to be related to treatment.

Dose descriptor:

LOAEL

Effect level:

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

Based on:

other: submission substance

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: submission substance

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: which were treatment-related or biological meaningful

Details on embryotoxic / teratogenic effects:
No treatment related effects on fetal ossification variations, external, soft tissue or skeletal malformations were found.
The malformations observed among control and treated groups included:
anasarca, localized edema, omphalocele, bent tail, microphthalmia, interventricular septal defect, vestigial uterine horn, bent ribs, interrupted ossification of ribs, bent scapula and bent limb bones. The malformations were considered incidental and occurred in no more than two fetuses per group. Although the mid-dose group was observed to have more litters with malformations than the control group, this was not considered meaningful as similar increases did not occur in the high-dose group.
Moreover all the above mentioned anomalies found could also be seen in the data of historical controls.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Oral administration of the test item to the rat at doses of 0, 50, 100 or 250 mg/kg body weight (corresponding to 0, 40, 80, and 200 mg submission substance/kg bw) from gestation day 6 - 15 caused early deaths as well respiratory rales in the maternal animals. In the foetuses no treatment related effects on foetal ossification variations, external, soft tissue or skeletal malformations could be found. With regard to the present study a No Observed Adverse Effect Level (NOAEL) for maternal toxicity could not be established. The respective maternal LOAEL (including effects on reproduction and fertility) in this study is 40 mg/kg/day. Based on the lack of any effects concerning the foetal development the developmental NOAEL of this study is 200 mg/kg/day.

Executive summary:

Groups of 25 mated female CD rats received the test material by oral gavage once daily at the dose levels of 0, 50, 100 or 250 mg/kg body weight (corresponding to 0, 40, 80, and 200 mg submission substance/kg bw) from gestation day 6 to 15 (day 0 = day the vaginal plug was observed) and were sacrificed an day 20 of pregnancy (equivalent to OECD 414, GLP).

Four rats in the 200 mg/kg/day dosage group, five rats in the 80 mg/kg bw/day dosage group and one rat in the 40 mg/kg bw/day dosage group died on study during gestation days 7 to 12, 7 to 20, and day 11, respectively. The cause of death could not be determined for any of these animals. One rat each in the 80 and 200 mg/kg/day dosage groups were sacrificed in extremis. Survival was 100% in the control group. Breathing difficulties occurred in all treated groups and were considered a result of treatment. Necropsy observations were comparable between the control group and all treated groups. Body weight gains in the all treated groups were reduced when compared to the control group values during the early treatment period (GD 6 to 9). However, a high degree of variability precluded the assessment of any meaningful relation to the test article at any specific interval.

Slightly increased postimplantation losses coupled with a decrease in corpora lutea were observed in the 40 and 80 mg/kg bw/day dosage groups when compared to the control group values. Consequently, the mean viable foetuses values for these groups were lower than the control group value. As these parameter values for the 200 mg/kg bw/day dosage group were comparable to the control group values, the findings in the 40 and 80 mg/kg bw/day dosage groups were not considered treatment-related. The mean foetal body weight and foetal sex distribution values for all treated groups were comparable to the group values.

There were no biologically meaningful differences in the incidence of foetal malformations and developmental variations between the control and treated groups.

Under the tested conditions the submission substance, did not produce a developmental/teratogenic effect (NOEAL (developmental) = 200 mg/kg bw/day). Nevertheless there was maternal toxicity seen throughout the study therefore resulting in a LOAEL (maternal) of 40 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two studies are available (full teratogenicity study and range finding study (Klimisch score 2 or 3 respectively)) - as no effecs on foetal development were observed the NOAEL presented is the highest dose tested in the full teratogenicity with the submission substance.

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

Groups of 25 mated female CD rats received the test material by oral gavage once daily at the dose levels of 0, 50, 100 or 250 mg/kg body weight (corresponding to 0, 40, 80, and 200 mg submission substance/kg bw) from gestation day 6 to 15(day 0 = day the vaginal plug was observed) and were sacrificed an day 20 of pregnancy.

Four rats in the 200 mg/kg bw/day dosage group, five rats in the 80 mg/kg bw/day dosage group and one rat in the 40 mg/kg/day dosage group died on study during gestation days 7 to 12, 7 to 20, and day 11, respectively. The cause of death could not be determined for any of these animals. One rat each in the 80 and 200 mg/kg bw/day dosage groups were sacrificed in extremis. Survival was 100% in the control group. Breathing difficulties occurred in all treated groups and were considered a result of treatment. Necropsy observations were comparable between the control group and all treated groups. Body weight gains in the all treated groups were reduced when compared to the control group values during the early treatment period (GD 6 to 9). However, a high degree of variability precluded the assessment of any meaningful relation to the test article at any specific interval.

Slightly increased postimplantation losses coupled with a decrease in corpora lutea were observed in the 40 and 80 mg/kg bw/day dosage groups when compared to the control group values. Consequently, the mean viable foetuses values for these groups were lower than the control group value.

Effect in low dose group is based on findings in only one out of 25 animals which had 11 late resorptions while all the other animals in the group had no late resorptions. In the mid dose group two out of 25 animals showed either 13 or 11 late resorptions while all other animals showed no increase concerning this read-out compared to controls.

As the reproduction parameter values for the 200 mg/kg bw/day dosage group were comparable to the control group values and no dose-related trend as the 40 mg/kg bw/day appeared to be marginally more affected than the 80 mg/kg bw/day group (at least for the decrease in mean corpora lutea number, total implantations and numbers of viable foetuses), the findings in the low and mid dosage group were not considered treatment-related.

The mean foetal body weight and foetal sex distribution values for all treated groups were comparable to the group values.

There were no biologically meaningful differences in the incidence of foetal malformations and developmental variations between the control and treated groups.

Under the tested conditions the submission substance, did not produce a developmental/teratogenic effect (NOAEL (developmental) = 200 mg/kg bw/day). Nevertheless there was maternal toxicity seen throughout the study therefore resulting in a LOAEL (maternal) of 40 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one teratogenicity study available (Klimisch score 2)

Justification for selection of Effect on developmental toxicity: via inhalation route:
Not rquired as there are no adverse effects on reproductive organs or tissues in repeated dose toxicity studies (28 day or 90 day study; one and two generation study) and no indications for reproductive toxic effects in the prenatal developmental oral toxicity study for the submission substance with rats. Moreover there are no effects on reproduction and no malformation or averse effects on pups were identified in a two generation study with rats (oral: feed) as well as mating trials following a subchronic onhalation exposure performed with rats or mice.

Justification for selection of Effect on developmental toxicity: via dermal route:
Not rquired as there are no adverse effects on reproductive organs or tissues in repeated dose toxicity studies (28 day or 90 day study; one and two generation study) and no indications for reproductive toxic effects in the prenatal developmental oral toxicity study for the submission substance with rats. Moreover there are no effects on reproduction and no malformation or averse effects on pups were identified in a two generation study with rats (oral: feed) as well as mating trials following a subchronic onhalation exposure performed with rats or mice.

Justification for classification or non-classification

Based on the above presented data no classification for reproductive and developmental effects is necessary according to Regulation (EC) No 1272/2008 and Council Directive 67/548/EEC.