1,6-dichlorohexane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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:

2012-05-14 to 2012-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study OECD guideline was followed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted July 27, 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl: CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 59 days
- Weight at study initiation: (P) Males: 318.0 to 359.4 g; Females: 182.4 to 217.6 g
- Housing: the males and females (F0 generation) were kept singly in MAKROLON cages (type III plus)
- Diet: ad libitum (Commercial ssniff R-Z V1324)
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Photoperiod (hrs dark / hrs light): darkened for periods of 12 hours

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in the vehicle corn oil to the appropriate concentrations. The test item formulations were freshly prepared on each administration day. Immediately after preparation of the test item-vehicle mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature: 3 samples/dose level group


VEHICLE
- Concentration in vehicle: 0, 70, 210, 630 mg/kg b.w./day
- Amount of vehicle (if gavage): 2 mL vehicle/kg b.w./day

Details on mating procedure:

- M/F ratio per cage: 1 male /1 female
- Length of cohabitation:
- Proof of pregnancy: Each day: avaginal plug; presence of sperm
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures samples of approx. 10 mL were taken at the following time points and stored at -20°C or colder until analysis.
Start of treatment period:
- Concentration and stability: Immediately after preparation of the test item-vehicle mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature: 3 samples/dose level group (groups 2 to 4).
- Homogeneity: At start of administration, during (middle) administration and before administration to the last animal of each dose level group: 3 samples/dose level group (groups 2 to 4).
Number of samples: 2 x 9
End of treatment period:
- Concentration: During treatment with the test item always before administration to the last animal/dose level group: 1 sample/dose level group (groups 2 to 4).
Number of samples: 3

Duration of treatment / exposure:

Males: 35 days
Females: 59 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 7, 210, 630 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels had been selected by the Sponsor based on the results of a 14-day dose-range-finding study (LPT Study No. 28420; see IUCLID-section: 7.5.1). Treatment with 1,6-Dichlorohexane caused reduced motility - in a dose-related way - in a few to all male and female animals from a dose level of 100 mg/kg b.w./day on-wards.
Furthermore, ptosis, reduced body weight and food consumption were observed from a dose level of 300 mg/kg b.w./day onwards in all animals.
In addition to the afore-mentioned intolerance reactions, treatment with 1000 mg test item/kg b.w./day caused ataxia and slight to moderate salivation. One of five male high dose rats and one of five female high dose rats died prematurely on test day 6.
Necropsy of the prematurely deceased animals revealed pulmonal lesions and/or gastrointestinal changes.

Positive control:

no positive control

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 post-partum

OTHER:
- Food and drinking water consumption: The quantity of food left by individual animals was recorded on a weekly basis through-out the experimental period and, for the females, daily during gestation and on day 4 post-partum.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, histopathology

Litter observations:

The duration of gestation was recorded and calculated from day 0 of pregnancy. As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post partum. Any abnormal behaviour of the offspring was recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The male animals were sacrificed after a minimum total dosing period of 28 days if no longer needed for further mating.
- Maternal animals: Dams with offspring were sacrificed on day 4 post-partum. Females showing no evidence of copulation were sacrificed 24 days after the last day of the mating period.

GROSS NECROPSY
At the time of sacrifice, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded in the female adult animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI .


HISTOPATHOLOGY / ORGAN WEIGHTS
The testes and epididymides of all male adult animals were weighed.
The ovaries (2), testicles (2), epididymis (2), accessory sex organs (coagulating gland, preputial gland, prostate, seminal vesicle, uterus (incl. cervix and oviducts), vagina) and all organs showing macroscopic lesions of all adult animals were preserved. The testes and epididymides were preserved in Bouin’s fixative; the remaining tissues were pre-served in 7% buffered formalin.
Detailed histopathologic examination was performed on the ovaries, testes and epidi-dymides (with special emphasis on the qualitative stages of spermatogenesis and histo-pathology of intestitial testicular structure) of the adult animals of the highest dose group and the control group following haematoxylin-eosin and PAS staining.

Postmortem examinations (offspring):

SACRIFICE
Dead pups and pups sacrificed at day 4 post-partum were carefully examined externally for gross abnormalities.

Statistics:

The test item-treated groups (2 to 4) were compared with the control group (1).
The following statistical methods were used:
-STUDENT's t-test: All numerical functional tests (p ≤ 0.01)
- Multiple t-test based on DUNNETT, C. W. (New tables for multiple Comparisons with a control Biometrics, 482-491 (Sept 1964)): Body weight / Food consumption / Relative organ weights (p ≤ 0.01)

For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance was p ≤ 0.01.
Exact test of R. A. FISHER: Histopathology, if applicable (p ≤ 0.05)

For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01) were employed.

Reproductive indices:

For each group the gestation index was determined:
- Fertility index female [%] = (number of pregnant rats/number of females used)x100
- Gestation Index = (number of litters with live pups/number pregnant)x100

Offspring viability indices:

For each litter and group the following indices were determined:
- Birth Index = (Total number of pups born (live +dead)/Number of implantation scars) x100
- Live birth Index = (Number of pups born alive on day 0/1 /Total number born (live + dead))x100
- Viability Index = (number of pups alive on day 4/number of pups live on day 0/1)x100
- Pre-implantation loss [%] = (corpora lutea - implantations/corpora lutea)x100
- Post-implantation loss [%] =(implantations - number of pups born alive/implantations) x100

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Mortality: None of the male or female rats treated with 70, 210 or 630 mg test item/kg b.w./day died prematurely during the course of the study.
Clinical signs:
-Male: At 210 mg test item/kg b.w./day slightly or moderately reduced motility was noted in 3 of 10 male rats and slightly increased salivation was noted in one further male on one or two test days. One male revealed piloerection for a period of seven days. At 630 mg test item/kg b.w./day 10 of 10 animals revealed reduced motility and 9 of 10 revealed ptosis during the first test week. Thereafter the number of affected animals declined rapidly. During the further course of study, piloerection and slightly or moderately increased salivation were observed in 6 or 7 males on several test days.
- Female: The intermediate dose level of 210 mg test item/kg b.w./day caused slightly or moderately increased salivation and piloerection in 2 of 10 females each on one or two days during the pre-mating, mating, gestation or lactation period. Reduced motility and ptosis were noted in 10 of 10 animals treated with 630 mg test item/kg b.w./day during the first test week. Thereafter, reduced motility was only noted in three animals during the gestation period.
Piloerection and slightly or moderately increased salivation were partly noted in all animals during different time periods lasting from one day to more than half of study duration.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Male: From 210 mg test item/kg b.w./day onwards, a dose-dependent reduction in body weight and body weight gain was noted from test day 8 until termination of the study. A statistically significant reduction compared to the control was noted for the intermediate dose males (210 mg test item/kg b.w./ day) on test days 15 and 36 (at p ≤ 0.05) and for the high dose group (630 mg test item/kg b.w./day) at test day 8, 15, 22, 29 and 36 (p ≤ 0.01). The body weight gain was statistically significant reduced in the groups treated with 210 mg test item/kg b.w./day (at p ≤ 0.05 or p ≤ 0.01) or 630 mg test item/kg b.w./day (at p ≤ 0.01) at all time points.
- Female: A temporary reduction (p ≤ 0.05) in body weight gain was noted during the first test week in the highest treatment group (630 mg test item/kg b.w./day). A more pronounced and dose-related reduction in body weight was noted for the female rats on lactation day 4, being statistically significant compared to the control (p ≤ 0.01) at 210 and 630 mg test item/kg b.w./day. The body weight gain was statistically significant decreased on lactation day 4 (p ≤ 0.05 or p ≤ 0.01) at all tested dose levels (70, 210 or 630 mg test item/kg b.w./day).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Pre-coital time: No test item-related influence was noted.
- Gestation length: No test item-related influence was noted.
- Evaluation of reproduction parameters of the dams: There were no test item-related differences between the control group and the animals treated with 70 or 210 mg test item/ kg b.w./day in the number of corpora lutea, implantation sites, in the number and sex of pups, runts or malformed pups. No test item-related changes were noted in the values calculated for the gestation length, the birth index and the live birth index. No test item-related increases were noted for the pre- and post-implantation loss. At 630 mg test item/kg b.w./day, a slightly increased post-implantation loss of 17.5% was noted (control: 9.1%). A statistically significant reduction compared to the control was noted in the live birth index with 94.5% (control 100%).


FOOD AND WATER CONSUMPTION:
-Male: A statistically significant reduction in food consumption compared to the control (at p ≤ 0.01) was noted in the groups treated with 210 or 630 mg test item/kg b.w./day during the first test week. No test item-related influence was noted on the drinking water consumption (visual appraisal) at any of the tested dose levels.
- Female: A statistically significant reduction in food consumption compared to the control (at p ≤ 0.01) was noted at 210 or 630 mg test item/kg b.w./day during the first test week (at p ≤ 0.01) and at all tested dose levels (70, 210 or 630 mg test item/kg b.w./day) during the lactation period (p ≤ 0.05 or p ≤ 0.01).
An increased drinking water consumption was noted in 4 of 10 high dose animals (630 mg test item/kg b.w./day) during the pre-mating, mating and gestation period.

ORGAN WEIGHTS
No test item-related influences were noted for testes and epididymides of the male rats.

GROSS PATHOLOGY:
- Body weight at autopsy: From 210 mg test item/kg b.w./day onwards, a dose-dependent reduction was noted for the body weight at autopsy in male and female rats, resulting in a statistically significant reduction for both sexes at the high dose level (630 mg test item/kg b.w./day).
- Macroscopic post mortem findings: Macroscopic inspection at necropsy revealed no test item-related changes in the organs or tissues of male or female rats after treatment with either 70, 210 or 630 mg test item/kg b.w./day.

HISTOPATHOLOGY: The histomorphological examination of the reproductive organs testes, epididymides and ovaries after treatment with 70, 210 or 630 mg test item/kg b.w./day did not reveal any morphological lesions which are considered to be related to the test item.

Dose descriptor:

NOAEL

Effect level:

210 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

MORTALITY (OFFSPRING):
- Mortality: At 630 mg test item/kg b.w./day, the viability index of the pups was statistically significant (p <= 0.01) reduced to 74.1% in comparison to the control group (98.6%) due to the high incidence of cannibalised and prematurely deceased pups.
- Body weight At 210 and 630 mg test item/kg b.w./day, a dose-dependent reduction in mean litter weight was noted on lactation day 4, being statistically significant at p <= 0.01 compared to the control. A statistically significant reduction in total litter weight was noted for the high dose group (630 mg test item/kg b.w./day) on lactation days 1 and 4.
- Terminal examinations: External examinations at dissection revealed no external abnormalities in any of the pups examined.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

70 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: From a dose level of 210 mg test item/kg b.w./day onwards, a dose-dependent reduction was noted for the body weight of pups.

Reproductive effects observed:

not specified

Conclusions:

The following no-observed-effect levels (NOAEL) of 1,6 dichlorohexane were noted:
The NOAEL (no-observed-adverse-effect level) on the F0-generation was 210 mg/kg b.w./day, p.o..
The NOAEL (no-observed-adverse-effect level) of reproductive toxicity was 210 mg/kg b.w./day, p.o..
The NOAEL (no-observed-adverse-effect level) of the F1 offspring (pups) was 70 mg/kg b.w./day, p.o.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

70 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study
OECD guideline was followed (Klimisch 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproduction/developmental toxicity screening test was performed with 1,6-dichlorohexane in 80 rats (in four groups) according to OECD guideline no. 421. The vehicle was corn oil. The test item was administered orally to rats at dose levels of 70, 210 and 630 mg/kg bw/day during the pre-mating, mating and post-mating periods to parental males as well as during the pre-mating, mating, gestation and lactation periods until day 3 post-partum to parental female animals.

At 210 mg 1,6 -dichlorohexane/kg bw/day, reduced motility, piloerection and/or slightly or moderately increased salivation were temporarily noted in a few male or female rats. In addition, reductions were noted for body weight and food consumption.

Treatment with 630 mg 1,6 -dichlorohexane/kg bw/day caused - in relation to the dose - more distinct signs of toxicity in the form of reduced motility, ptosis and increased salivation in several to all rats of both sexes, piloerection, ataxia or increased drinking water conumption in one or few male or female animals as well as reduced body weight and food intake.

None of the parental animals died prematurely. Macroscopic inspection did not reveal any test item-related changes at necropsy.

No test item-related changes were noted for the weights of the testes and epididymides. Histopathological examination of the reproduction organs did not reveal any test item-related changes. No test item-related influence was noted on the qualitative sperm staging.

No test item-related influence was noted onmating behaviour, fertility, implantation, the gestation length or the birth index up to a dose level of 210 mg 1,6 -dichlorohexane/kg b.w./day.

At the materno-toxic dose level of 630 mg 1,6 -dichlorohexane/kg bw/day, a slightly increased post-implantation lossand a statistically significantly reduced live birth index were noted due to an increased incidence of stillbirths.

No test item-related influence was noted up to a dose level of 70 mg 1,6 -dichlorohexane/kg bw/day on the growth and development of the offspring from conception until sacrifice on day 4 post-partum.

From a dose level of 210 mg 1,6 -dichlorohexane/kg bw/day onwards, a dose-dependent reduction was noted for the body weight of pups.

An adverse effect on the development of the pups was noted at the materno-toxic dose level of 630 mg 1,6 -dichlorohexane/kg bw/day, indicated by a reduced mean and total litter weight and a reduced survival rate of the pups.

The NOAEL (no-observed-adverse-effect level) on the F0-generation was 210 mg/kg bw/day, p.o..

The NOAEL (no-observed-adverse-effect level) of reproductive toxicity was 210 mg/kg bw/day, p.o..

The NOAEL (no-observed-adverse-effect level) of the F1 offspring (pups) was 70 mg/kg bw/day, p.o.

1,6 -Dichlorohexane is manufactured/formulated/used at industrial sites in a closed system. Under the strict conditions implemented no repeated exposure of worker is expected. The substance is not intended for consumer use. Furthermore, in the available combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening test no external abnormalities were observed in any of the pups examined. Therefore no developmental toxicity/teratogenicity study is required due to animal welfare reasons. The available OECD422 study is sufficient for risk assessment.

Short description of key information:
A reproduction/developmental toxicity screening test was performed with 1,6-dichlorohexane according to OECD guideline no. 421.
The following no-observed-effect levels (NOAEL) of the test item were noted:
The NOAEL (no-observed-adverse-effect level) on the F0-generation was 210 mg/kg bw/day, p.o..
The NOAEL (no-observed-adverse-effect level) of reproductive toxicity was 210 mg/kg bw/day, p.o..
The NOAEL (no-observed-adverse-effect level) of the F1 offspring (pups) was 70 mg/kg bw/day, p.o.

Justification for selection of Effect on fertility via oral route:
Only one key study is available.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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

Justification for selection of Effect on developmental toxicity: via oral route:
A teratogenicity /pre-natal developmental toxicity study according to OECD 414 is proposed.

Justification for classification or non-classification

Based on the results of the reproduction/developmental toxicity screening study, 1,6 -dichlorohexane is not classified according to EU Directive 67/548/EEC or EU Regulation (EC) No 1272/2008.