Carbamic acid ester

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study under GLP with limited number of investigations. The information in the report is limited to what is included in the summary

Data source

Reference

Reference Type:

other: study report in the public domain

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mice were exposed to the substance for 13 weeks and thereafter selected for mating and reproduction testing. Exposure was continued during the mating period and pregnant females were allowed to deliver and nurse their pups until day 21 of lactation. The protocol resembles that of OECD 422.

GLP compliance:

yes

Remarks:

no QA statement and/or study director statement included in the report, but GLP is claimed

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

aerosol dispenser: not specified

Remarks:

migrated information: aerosol

Details on test material:

The test material is an anlogue of the expected metabolite of the substance

Identity: Hexane diamine (identified by infrared spectroscopy)
Lot: PT-031985
Purity: 70.9% (purchased as a 70% aqueous solutionfrom E. I. DuPont de Nemours and Company, Inc. (Wilmington, DE))
Stability: stable for 4 months
Storage: at room temperature in amber or foil-wrapped bottles

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6-7 weeks
- Average weight at study initiation: males 20.6-24.8 g; females 17.9-19.8 g (no additional information on animals used for mating)
- Housing: individually
- Diet: Pelleted NIH-07 feed (Zeigler Brothers, Inc., Gardners, PA) ad libitum during non-exposure
- Water: ad libitum during non-exposure
- Acclimation period: 11-14 days

ENVIRONMENTAL CONDITIONS (non-exposure)
- Temperature (°C): ca 22 °C
- Humidity (%): 50% ± 15%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

For the inhalation studies, 1,6-hexanediamine was converted to 1,6-hexanediamine dihydrochloride (HDDC) by acidification with concentrated hydrochloric acid under a stream of nitrogen. The final pH was adjusted within the range of 4.5 to 5.5 before storage and again before use in the inhalation chambers.
The 70% aqueous HDDC solution was placed in a 9-liter glass reservoir and pressurized with N2 gas. HDDC was delivered to 5 Sonimist Ultrasonic Spray Nozzles (Model HS6002, Heat Systems-Ultrasonics, Inc., Farmingdale, NY) by a positive displacement metering pump. Up to this point, stainless steel lines carried the test substance. The nebulizer reservoir was kept in a separate exposure chamber (H-1000, Hazelton Systems, Inc., Aberdeen, MD). This chamber served as a mixing plenum where large droplets and nonnebulized liquid were impacted or sedimented out of the test atmosphere before the aerosol was delivered to the inhalation chambers. The HDDC aerosol was mixed with compressed breathing air that had been filtered through an ENMET (ENMET Air Filtration Panel, Model AFP-82, Enmet Co., Ann Arbor, MI) and supplied at 50 psi to generate an aerosol at a concentration equal to the highest exposure concentration. The resulting aerosol was transported to the inhalation chambers through a manifold constructed of 3-inch diameter PVC tubing. At each chamber, a metered amount of aerosol was removed from the manifold and mixed with the appropriate amount of HEPA/charcoal-filtered room air to obtain the desired test concentration, then delivered to the inhalation chamber. After exiting the chambers, the test atmospheres were delivered to a common duct and cleansed of the test substance by a Mystaire HS-7CM scrubber (Heat Systems Ultrasonics).


Method of holding animals in test chamber: individually in compartments of multi compartment wire mesh cages, during exposure in stainless steel andglass exposure chambers of 2 m3 volume, with 15 air changes per hour (500 L/min). During inhalation exposures, chambers were maintained at 22°C to 25°C and 70% to 80% relative humidity.

TEST ATMOSPHERE
- Brief description of analytical method used: forward light scatter with RAM-S real-time aerosol monitors (GCA Corporation,Technology Division, Bedford, MA) and gravimetric analyses of filter samples collected from each exposure chamber
- Samples taken from breathing zone: no, 6 RAM-S readings and 3 gravimetric samples were taken from each exposure chamber on each day of exposure.

Details on mating procedure:

- M/F ratio per cage: 1 male and 2 females
- Length of cohabitation: maximum 10 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
- No replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: exposure for 13 weeks including the mating period, but no exposure of females during gestation and lactation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Twice monthly during the 13-week studies, glass fiber filter samples from each chamber were analyzed by gas chromatography with flame ionization detection for total hexanediamine, using the technique supplied by Midwest Research Institute. Measured concentrations of HDDC in the exposure chambers were within 6% of the target concentrations in all samples.
Spatial homogeneity of the aerosol within the exposure chambers was determined using the calibrated RAM-S monitors. Chamber concentrations were measured at 12 points within each chamber and then were compared to a fixed reference point. Time spans required to reach stable concentrations after start up and to reach background concentrations at the end of exposure were determined by taking measurements of aerosol concentrations every 60 seconds. The time span required after start up to reach 90% of the target concentration was identified as the T90; the time span required after the end of the exposure period to reach 10% of the target concentration was identified as the T10.
Triplicate particle size measurements were obtained for each exposure chamber once in the first week and monthly thereafter, using an APS 3300 aerodynamic particle sizer (TSI, Inc., Minneapolis, MN). In addition, a CFM Ambient Impactor (Flow Sensor, McLean, VA) cascade impactor was used to determine the particle size distribution in the highest exposure level chamber once during the 13-week studies. The mass median aerodynamic diameter values for each chamber ranged from 1.62 to 1.72 microns, with a geometric standard deviation of 1.52 to 1.53. All control chamber respirable mass concentration values were less than 0.005 mg/m3.

Duration of treatment / exposure:

13 weeks (including mating period)

Frequency of treatment:

6 hours(+ T90 (=30 min))/day, 5 days per week

Details on study schedule:

Mating was between exposure day 68 and 80 of the study (only weekdays counted)
Males were terminated immediately thereafter. Females were checked upto 23 days after mating for signs of copulation and terminated thereafter if non-pregnant.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 50 and 160 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

20 males and 40 females

Control animals:

yes

Details on study design:

no data

Positive control:

none included

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes no details

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT (GAIN): Yes
- Time schedule for examinations: both sexes at start; males at the end of the mating period; females at day 0 and 20 of gestation], day 0, 5, 14 and 21 of lactation

FOOD CONSUMPTION: No data

Oestrous cyclicity (parental animals):

see under repeated dose

Sperm parameters (parental animals):

see under repeated dose

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain (BW day 0, 5, 14 and 21 of lactation), physical abnormalities

Postmortem examinations (parental animals):

males: none
females: necropsy only on non-pregnant females: uterus examination for signs of pregnancy (includes ammonium sulfide staining to verify implementation)

Postmortem examinations (offspring):

none

Statistics:

Continuous, quantitative data, such as body weights, were analyzed by Dunnett's t-test for multiple comparisons to a single control group. Discrete, counting data, such as litter counts, were analyzed by the Mann-Whitney U nonparametric test. Percentage data, such as the fertility and survival indices, were analyzed by the Chi Square test.

Reproductive indices:

no data

Offspring viability indices:

no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not examined

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

MORTALITY (PARENTAL ANIMALS): 3 females at 16 mg/m3, 1 male and 1 female at 50 mg/m3

BODY WEIGHT (PARENTAL ANIMALS): no treatment related effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
pregnancy rate: 35, 32, 33 and 35 at 0, 16, 50 and 160 mg/m3
gestation length: significantly increased at 50 and 160 mg/m3 (no biological relevance)

Effect levels (P0)

Dose descriptor:

NOAEC

Effect level:

5 mg/L air (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see repeated dose toxicity study

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING): no treatment related effects

LITTER SIZE: no treatment related effects

SEX RATIO: no treatment related effects

CLINICAL SIGNS (OFFSPRING); no treatment related effects

BODY WEIGHT (OFFSPRING): no treatment related effects (significantly decreased in pups at 160 mg/m3 on day (14 and) 21 of lacatation)

Effect levels (F1)

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

160 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see study

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Mean Body Weights and Length of Gestation for Female B6C3F₁ Mice in the Mating Trial Study og 1,6 -hexanediamine Dihydrochloride

	0 mg/m3	16 mg/m3	50 mg/m3	160 mg/m3
Dam Weight During Gestation^1	22 25.6 ± 0.3 44.3 ± 0.8 18.7 ± 0.8 -	23 25.6 ± 0.4 44.4 ± 0.8 18.8 ± 0.8 100	27 27.2 ± 0.5* 43.4 ± 0.8 16.2 ± 0.9 98	24 26.2 ± 0.3 44.2 ± 0.5 18.0 ± 0.5 100
Lenght of Gestation^1	30 17.68 ± 0.10	30 18.00 ± 0.12	31 18.11 ± 0.09**	31 18.11 ± 0.08**
Dam Weight During Lactation^1   Lactation day 0     Lactation day 5     Lactation day 14     Lactation day 21	35 31.4 ± 0.3   34 35.3 ± 0.4   34 39.7 ± 0.5   34 34.8 ± 0.5	32 32.0 ± 0.4   32 35.7 ± 0.5   32 39.6 ± 0.6   32 34.2 ± 0.5	33 32.5 ± 0.4   33 35.3 ± 0.4   33 38.9 ± 0.6   33 34.7 ± 0.6	35 31.8 ± 0.3   35 34.9 ± 0.4   35 38.8 ± 0.6   35 33.9 ± 0.61

  ¹Data presented as mean ± standard deviation. Differences from the control group were evaluated by Williams’ or Dunnett’s test (weight) and Shirley’s test (gestation length)

* Significantly different (P<0.05) from the control group by Williams’ test.

** Significantly different (P<0.01) from the control group by Shirley’s test.

 

Survival, Sex Distribution and Mean Body Weights of B6C3F₁ mouse Pups in the Mating trial Study of 1,6 -Hexanediamine Dihydrochloride.

	0 mg/m3	16 mg/m3	50 mg/m3	160 mg/m3
Day 0	35 9.11 ± 0.38 8.86 ± 0.37 53.33 ± 3.51 1.37 ± 0.03	34 9.00± 0.44 8.53 ± 0.53 50.56 ± 3.56^2 1.39± 0.03^2	33 8.73± 0.46 8.61 ± 0.45 53.18 ± 2.11 1.41 ± 0.04	35 9.57± 0.25 9.37 ± 0.24 50.56 ± 3.25 1.37 ± 0.02
Day 5	35 8.54     55.94 ± 3.53^3 3.57 ± 0.08^3	34 8.44 ± 0.52 50.79 ± 3.58^2 3.56 ± 0.08^2	33 8.58 ± 0.46 53.28 ± 2.11 1.41 ± 0.04	35 9.37 ± 0.24 50.32 ± 3.29 3.55 ± 0.06
Day 14	35 8.51 ± 0.44 56.43 ± 3.51^3 8.19 ± 0.21^3	34 8.41 ± 0.52 50.80 ± 3.58^2 7.95 ± 0.15^2	33 8.58 ± 0.46 53.28 ± 2.51 8.25 ± 0.27	35 9.34 ± 0.24 50.08 ± 3.33 7.63 ± 0.16
Day 21	35 8.46 ± 0.43 56.15 ± 3.58^3 10.94 ± 0.24^3	34 8.41 ± 0.52 51.11 ± 3.57^2 10.68 ± 0.17^2	33 8.58 ± 0.46 53.28 ± 2.51 10.93 ± 0.29	35 9.31 ± 0.24 50.03 ± 3.26 10.21 ± 0.19*

1 Data presented as mean ± standard deviation. Differences from the control group for percent of live male pups, litter size, and number of pups born alive are not significant by Dunn’s test. The significance of differences in pup weights between dosed and control groups was evaluated by Dunnett’s or William’s test.

2 n=32

3 n=34

* Significantly different (P<0.05) from the control group by William’s test

Applicant's summary and conclusion

Conclusions:

There was no effect on male or female fertility, body weight (gain), gestation length and litter size. The reduced pupweights during part of the lactation period are considered not biologically relevant. No effects were noted on neonatal survival, sex ratios of pups and pup morphology in mice exposed to the substance.

Executive summary:

Male (n=20) and female (n=40) mice were exposed to the substance at 0, 16, 50 and 160 mg/m3 for 13 weeks (6 h/day, 5 d/wk). Animals were mated (1 male/2 females) and females showing signs of copulation and pregnancy were further evaluated during gestation and lactation (until day 21 of lactation). Males and non-pregnant females were terminated either immediately after mating (males) or after 23 days (non-pregnant females). The pups were checked for neonatal survival, weights, sex ratios and morphology. No treatment related mortality, effects on body weight(gain) or effects on reproductive performnce were observed in parental animals. No adverse effects were found in the offspring.