6-chlorohexan-1-ol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Description of key information

The reproductive and developmental toxicity of 6-chlorohexan-1-ol, has been investigated in Wistar Han rats in a repeated dose, reproductive and developmental screening study conducted according to OECD TG 422. In the study, three respective groups of male and female rats (10 rats per group) were administered the substance daily (7 days/week) at concentrations of 25, 80 and 250 mg/kg bw/day by oral gavage administration for at least 28 days.  Control groups received a reverse osmosis water vehicle. No adverse parental toxicity was observed up to 80 mg/kg/day. One female at 250 mg/kg/day was sacrificed on Day 0 post-coitum with severe body weight loss, hunched posture, piloerection and uncoordinated movements. There were no findings of one animal only, but a relation to treatment with the test item could not be excluded. This premature decedent was regarded adverse. Female at 80 mg/kg/day was sacrificed on Day 23 post-coitum because of delivery difficulties. Decreased body weights and/or body weight gain were recorded at 250 mg/kg/day. In males body weights were lower from Day 8 of treatment onwards while in females reduced body weight gain was recorded during the lactation period, correlating with a decreased food.  Considering the magnitude of change these effects were regarded adverse. Thus, parental NOAEL was established at 80 mg/kg/day (based on a premature decedent, and adverse effects on body weight and food consumption). No treatment-related changes were noted in reproductive performance and any of the other reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). Thus, NOAEL was established at 250 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start:13 Oct 2020 Experimental Completion: 15 Dec 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Annex VIII Data Requirement

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many
regulatory authorities and there are ample experience and background data on this species
and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: males: 10-11 weeks; females: 13-14 weeks
- Weight at study initiation: 250-289 g for males and approximately 217 to 256 g for females
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): drinking water
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 °C
- Humidity (%): 40-70%
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

Supplier: Merck Specific gravity: 1.036

Details on exposure:

Oral gavage at a constant dose volume of 5 mL/kg

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20257580).
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was  10%.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20257580) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20257580.

Duration of treatment / exposure:

Males: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing,, through the pairing period and thereafter until the day before necropsy, for a minimum of 29 days.
Females that delivered were treated for 51-63 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-52 days.

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations: Males: weekly
Females: weekly from allocation to positive identification of mating and on Days 0, 4, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for haematology, coagulation and clinical chemistry were collected, at the end of treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (food deprivation)
- How many animals: 5 males and 5 females (with viable litters) of each group


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for haematology, coagulation and clinical chemistry were collected, at the end of treatment period
- Animals fasted: Yes (food deprivation)
- How many animals: 5 males and 5 females (with viable litters) of each group


PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: at termination, the timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days.
- Animals fasted: Not specified


URINALYSIS: Yes (only males)
- Time schedule for collection of urine: during the last week of treatment
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No
- Parameters checked in table [No.13] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the study, toward the end of treatment
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations:
testis weight, epididymis weight, daily sperm production, identification of the stages of the spermatogenic cycle

Litter observations:

MORTALITY
Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Pups showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.
CLINICAL OBSERVATIONS
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.
BODY WEIGHTS
Live pups were weighed individually on PND 1, 4, 7 and 13
SEX
Sex was externally determined for all pups on PND 1 and 4.
ANOGENITAL DISTANCE
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
AEROLA/NIPPLE RETENTION
All male pups in each litter were examined for the number of areola/nipples on PND 13.
CULLING
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible, from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Postmortem examinations (parental animals):

NECROPSY

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Blood of F0-animals (except for animals which were sacrificed in extremis or females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in
the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0- females were not fasted overnight. Blood of F1-animals was collected on PND 4 and PND 14-16. This was performed in the necropsy room. On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by
decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. As for Litter No. 62, the target volume of 0.5 mL was not reached by pooling two pups, a third surplus pup of the same litter was added.
On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). As for Litter No. 59 and 73 the target volume of 1.0 mL/pup was not reached, a separate blood sample was collected from another pup of the same litter and sex. Any incomplete blood sample was discarded. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure.
Representative samples of the tissues identified in the table below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated: Animal identification; Artery; aorta; Body cavity, Nasopharynx; Bone: marrow, femur, sternum, Brain (eight levels), Cervix, Epididymides, Esophagus, Eye, Gland: adrenal, coagulation, Harderian, lacrimal, mammary parathyroid, pituitary, prostate, salivary, seminal vesicle, thyroid; Gross lesions/masses; Gut-associated lymphoid tissue; Heart: Kidney; Large intestine: cecum, colon, rectum; Larynx; Liver; Lung; Lymph node (mandibular and mesenteric site); Muscle, skeletal; Nerve: optic, sciatic; Ovaries; Pancreas; Skin; Small intestine: duodenum, ileum, jejunum; Spinal cord; Spleen; Stomach; Testes; Thymus; Tongue; Trachea; Urinary bladder; Uterus; Vagina.

HISTOLOGY:
Selected animals and unscheduled deaths (sacrificed in extremis):Tissues identified above (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
Females with total litter loss: Mammary gland.
Remaining animals: Gross lesions/masses.
HISTOPATHOLOGY
All tissues as defined under Histology – F0-Generation were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported.
For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Postmortem examinations (offspring):

Recognizable fetuses of females that were euthanized in extremis (No. 66) were examined externally and sexed (both externally and internally). Live fetuses were euthanized by decapitation.
Pups that died or were euthanized before scheduled termination were examined externally and
sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the
presence of milk, if possible. If possible, defects or cause of death were evaluated.
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litte, and the thyroid from two pups per litter was preserved in 10% buffered formalin.

Statistics:

For continuous variables the significance of the differences amongst group means was
assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric
version of theWilliams test. The mean values, standard deviations and statistical
analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

Mating Index, Preicoital time; Fertility index; Gestation index; Duration of gestation

Offspring viability indices:

Post Implantation Survival Index;
Live Birth Index;
Viability Index;
Lactation Index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during daily detailed clinical observations up to 80 mg/kg/day.
At 250 mg/kg/day incidental breathing rales were recorded in 3/10 males and 2/10 females during the first three weeks of treatment. Females at 250 mg/kg/day also showed hunched posture (4/10) and uncoordinated movements (1/10) during this period. Piloerection was recorded in one female during Days 2-10 post-coitum. As clinical signs were transient, they were regarded not toxicologically relevant.
Salivation seen after dosing among animals of the 80 and 250 mg/kg/day dose groups starting at Day 21 of the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period (alopecia) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females were sacrificed for human reasons:
Female No. 66 (80 mg/kg/day) was sacrificed in extremis on Day 23 post-coitum because of delivery difficulties. Bleeding of the genital region was recorded on the day of sacrifice. At necropsy dead/autolytic fetuses were found in the uterus. No microscopic findings of note were recorded; all findings were regarded to be related to the pregnancy and incomplete delivery. These delivery problems in a single female at 80 mg/kg/day were regarded unrelated to treatment with the test item.
Female No. 71 (250 mg/kg/day) was sacrificed in extremis on treatment Day 18 (i.e. the day of mating) due to severe body weight loss (~ 9% in three days, recorded in study raw data) and clinical signs (hunched posture, piloerection and uncoordinated movements). There were no findings of note that could explain the poor condition of this animal.
One female of the 250 mg/kg/day group (No. 80) was euthanized on Lactation Day 2, as she had a total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes in body weights and body weight gain were observed up to
80 mg/kg/day.
Body weights and body weight gain in males at 250 mg/kg/day were decreased from Day 8 of treatment onwards (up to 10% lower than control for absolute body weight on Day 15 of the
mating period).
In females at 250 mg/kg/day decreased body weight gain was recorded during the lactation period (up to 60% lower than control, statistically significant on Day 13 of lactation only)
resulting in an absolute body weight of 4% lower than control on Lactation Day 13 (not statistically significant).
Any other changes in body weights and body weight gain were considered to be unrelated to treatment regarding the direction of change and absence of a dose-related trend.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded up to 250 mg/kg/day for males and up to 80 mg/kg/day for females.
In females at 250 mg/kg/day food consumption was reduced during the first two weeks of treatment (up to 19% lower than control for absolute food consumption, not statistically significant), which subsequently recovered. In addition, decreased food consumption (before and after correction for body weight) was recorded during Days 4-13 of the lactation period (up to 23% lower than control on Days 7-13 of lactation). This findings was regarded treatment-related.
In males at 250 mg/kg/day food consumption was reduced during the first two weeks of treatment (up to 14% lower than control for absolute food consumption, not statistically
significant), which subsequently recovered. As this finding was transient it was considered of no toxicological relevance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following test item-related changes in hematology parameters were noted in treated males or females. Relative differences in mean values as compared to the control group are
indicated between parentheses.
- Increased red blood cell counts in males at 80 and 250 mg/kg/day (1.05 and 1.08x of control, respectively) with corroborative increased hemoglobin (1.07 and 1.08x of control, respectively) and hematocrit (each 1.08x of control) levels.
- Decreased white blood cell counts in females at 25, 80 and 250 mg/kg/day (0.72, 0.59 and 0.53x of control, respectively) with corroborative decreased neutrophil (0.67, 0.65
and 0.53x of control, respectively), lymphocyte (0.73, 0.56 and 0.53x of control, respectively) and eosinophil (0.72, 0.40 and 0.28x of control, respectively) concentrations (not always statistically significant).
- Decreased reticulocyte levels in females at 250 mg/kg/day (0.62x of control).
- Decreased platelet counts in females at 25, 80 and 250 mg/kg/day (0.90, 0.82 and 0.77x of control, respectively; not always statistically significant).
Any other changes in hematology parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Coagulation
In males and females at 250 mg/kg/day prolonged prothrombin time (PT) was recorded (1.24 and 1.08x of control, respectively), which was considered test item-related.
Prolonged activated partial thromboplastin time (APTT) recorded in males at 25, 80 and 250 mg/kg/day was considered the result of slightly low control values3 and considered not related to treatment with the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following test item-related changes in clinical biochemistry parameters were noted in treated males or females. Relative differences in mean values as compared to the control group are indicated between parentheses:
-Increased liver enzyme activities in males at 25, 80 and 250 mg/kg/day, consisting of increased alanine aminotransferase (ALT, 1.51, 1.70 and 1.52x of control, respectively) and aspartate aminotransferase (AST, 1.14, 1.49 and 1.34x of control,respectively) activities (not statistically significant at the low dose).
-Increased bile acids in males and females at 250 mg/kg/day (BILEAC, 1.99 and 4.16xof control, respectively).
-Increased urea concentrations in males at 250 mg/kg/day (1.77x of control).
-Increased creatinine concentrations in males (1.22x of control) while decreased in females (0.71x of control) at 250 mg/kg/day.
-Decreased cholesterol levels in males at 80 and 250 mg/kg/day (0.66 and 0.55 of control, not statistically significant at the mid dose).
-Decreased total protein and albumin concentrations in females at 250 mg/kg/day (0.87 and 0.81x of control, respectively).
Decreased chloride levels in males at 250 mg/kg/day was considered not toxicologically relevant, considering the magnitude of change and as individual values were within the same range as control.
Increased potassium levels in males and decreased potassium levels in females were considered the result of slightly low or high control values4 and were regarded not related to treatment with the test item.
Any other changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Thyroid hormone analyses:
Serum levels of T4 and TSH in F0-males were decreased at 250 mg/kg/day (0.74 and 0.30x of control, respectively). No differences were recorded in females.

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were considered unaffected by treatment with the test item. Motor activity was considered not to be affected by treatment with the test item. Lower mean total movements and ambulations in males and females at 250 mg/kg/day (not statistically significant) were considered not toxicologically relevant as in general individual values were within the same range as controls and mean values remained within the historical control range. All groups showed a similar motor activity habituation profile with a decreasing trend in
activity over the duration of the test period.
Not dose related decrease (up to -21%) in landing foot splay measurement (second trial) performed toward the end of treatment was recorded in treated males when compared to control. Mean decrease in Group 2 males was significant, at statistical analysis. In all cases the changes were within the historical control data (HCD), observed in only one sex and then without toxicological relevance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Test item-related higher liver weights and lower spleen, prostate gland, seminal vesicles and thymus (females) weights were noted. For the higher thyroid gland (females) weights a test
item-relationship could not be excluded. Statistically significant, test item-related higher liver weights were recorded at 80 and 250 mg/kg/day in males (relative to body weight only) and in females (absolute and relative to body weight). The microscopic correlate for the higher liver weight at 250 mg/kg/day was diffuse hepatocellular hypertrophy in males and cytoplasmic rarefaction (glycogen storage) in females.
Statistically significant, test item-related lower spleen weights were noted in males at 250 mg/kg/day (absolute only) and in females at 80 and 250 mg/kg/day (absolute and/or
relative to body weight). The microscopic correlate in females was decreased lymphoid cellularity and reduced extramedullary hematopoiesis. For males there was no microscopic correlate.
Statistically significant, test item-related lower prostate gland weights (absolute and relative to body weight) and seminal vesicles weights (absolute only) were noted in males at 250 mg/kg/day. There was no microscopic correlate for these lower weights.
Statistically significant, test item-related lower thymus weights were recorded in females at 80 and 250 mg/kg/day (absolute and relative to body weight). The microscopic correlate was
decreased lymphoid cellularity.
Statistically significant higher thyroid gland weights were noted in females at 80 and 250 mg/kg/day (relative to body weight only), both at comparable magnitude and without microscopic correlate. This weight change was regarded possibly test item-related.
There were no other test item-related organ weight changes. Some organ weight differences in males at 250 mg/kg/day were statistically significant when compared to the control group
but were considered to be the result of a test item related effect on final body weight. These included lower absolute weights for heart, testes and epididymides and higher brain relative to body weights.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with
the test item. The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on
Day 1 was considered unaffected by treatment with the test item. Viability indices were 98, 99, 99 and 96% for the control, 25, 80 and 250 mg/kg/day groups, respectively.
Two pups of the control and 250 mg/kg/day group and one pup at 25 and 80 mg/kg/day each
were found missing on PND 2-3. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
One pup at 250 mg/kg/day was sacrificed in extremis on PND 1. In this pup missing anus and genital region were recorded. At the incidence observed, no toxicological relevance was attached to these findings.
Female No. 80 (at 250 mg/kg/day) had total litter loss on Day 2 of lactation. The single, missing pup in this litter was recorded pale and without milk at first litter check. Although at single incidence, a relation to treatment with the test item could not be excluded.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups were decreased at 250 mg/kg/day on PND 13 (21% lower than control
in both sexes), which was considered test item-related. A trend towards lower body weights was also apparent at 80 mg/kg/day (7 and 6% lower than control for males and females on PND 13, respectively, not statistically significant).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by
treatment with the test item.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment with the test item up to 250 mg/kg/day had no effect on areola/nipple retention. For
none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No relevant differences were observed in the weight of thyroid in treated pups, when
compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups surviving up to scheduled necropsy.

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Post-implantation survival index was decreased at 25, 80 and 250 mg/kg/day., both compared to the control group of this study and to the historical control data (mean 91%, P5-P95= 83-27% (n=133). The post implantation survival index in group 4 is only lowered due to the total litter loss of female 80. At necropsy a thickened uterus was observed. Post-implantation survival index (Total Number of Offspring born as a percentage of total number of uterine implantation sites) was 94, 79, 75 and 74% for the control, 25, 80 and 250 mg/kg bw/day groups, respectively.
In Groups 2 and 4 mean litter size was significantly lower than the mean number of implantation site. However, in Group 3 the lower post-implantation survival index was caused by Female 63 with implantation site only and Female No.66 which was sacrificed during delivery.
For Female No. 48 (control), the number of pups were slightly higher than the number of implantation. This phenomenon is observed from time to time and is cased by normal sorption of these areas during lactation. No toxicological relevance was attached to this finding in the current study.
Litter size was decreased in female at 25 and 250 mg/kg/day, which was considered test item-related. Liver litter sizes were 11.6, 9.1, 11.6 and 8.6 living pups/litter for the control, 25, 80. And 250 mg/kg bw/day groups, respectively.
The concurred control range of liver litter size is 8-14 living pups/litter. There are 4/9 and 2/8 females with litter size below the lower end of the concurrent control range in Groups 2 and 4, respectively. A clear dose-relation is not apparent.
Slightly longer duration of gestation at 250 mg/kg/day may be the result of the smaller litter size in this group and is therefore likely to be an indirect effect. Considering the magnitude of change this was regarded not adverse.
Body weights of pups were decreased at 80 and 250 mg/kg/day on PND 13. Considering the magnitude of change, this finding was regarded non-adverse at 80 mg/kg/day but adverse at250 mg/kg/day.
A trend towards lower body weights was also apparent at 80 mg/kg bw /day (7 an 6% lower than control for males and females on PND 13), respectively and not statistically significant)
No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

ca. 25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Conclusions:

In conclusion, based on the results of this combined 28-day repated dose toxicity study with the rreproduction/developmental toxicity screening test, the following NOAELS were estabilished for the test substance:

Parental NOAEL:

Executive summary:

The purpose of this study was to determine the potential toxic effects of the test substance when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 25, 80 and 250 mg/kg/day, based on the results of the Dose Range Finder.

The study design was as follows:

 

Group No.	Treatment	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	Vehicle	0	5	0	10	10
2	Test Item	25	5	5	10	10
3		80	5	15	10	10
4		250	5	50	10	10

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormone T4 and TSH (F0-animals), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

toxicity was observed up to 80 mg/kg/day.

One female at 250 mg/kg/day was sacrificed on Day 0 post-coitum with severe body weight loss, hunched posture, piloerection and uncoordinated movements. There were no findings of note that could explain the poor condition of this animal. Premature mortality was recorded in one animal only, but a relation to treatment with the test item could not be excluded. This premature decedent was regarded adverse.

One female at 80 mg/kg/day was sacrificed on Day 23 post-coitum because of delivery difficulties. Bleeding of the genital region was recorded on the day of sacrifice. At necropsy dead/autolytic fetuses were found in the uterus. No microscopic findings of note were recorded; all findings were regarded to be related to the pregnancy and incomplete delivery.

This was regarded unrelated to treatment with the test item.

Decreased body weights and/or body weight gain were recorded at 250 mg/kg/day. In males body weights were lower from Day 8 of treatment onwards while in females reduced body weight gain was recorded during the lactation period, correlating with a decreased food consumption. Considering the magnitude of change these effects were regarded adverse.

Non-adverse test item-related morphologic alterations were present in liver of males starting at 25 mg/kg/day (periportal hepatocellular vacuolation, peribiliary infiltrates and diffuse hepatocellular hypertrophy; higher weights and macroscopic accentuated lobular pattern and/or pale discoloration; higher ALT, AST and bile acid values) and liver of females starting at 80 mg/kg/day (cytoplasmic rarefaction; higher weights), thymus of females starting at 80 mg/kg/day (decreased lymphoid cellularity; lower weights and reduced size; lower lymphocyte counts) and at 250 mg/kg/day in prostate gland of males (increased severity of inflammatory cell infiltrates), bone marrow (femur and sternum) of both sexes (decreased cellularity), spleen females (decreased lymphoid cellularity, decreased extramedullary hematopoiesis; lower weights; lower white blood cell, lymphocyte, neutrophil, reticulocyte and eosinophil counts) and mesenteric lymph node females (decreased lymphoid cellularity).

In addition, lower spleen, prostate gland and seminal vesicles weights in males at 250 mg/kg/day, lower spleen weight in females at 80 mg/kg/day and higher thyroid gland weights in females at 80 and 250 mg/kg/day were noted, all without microscopic correlates and non-adverse.

Additional non-adverse changes in hematology and clinical biochemistry parameters comprised of: increased red blood cell, hemoglobin and hematocrit counts, increased urea and creatinine concentrations, decreased cholesterol levels (males) and decreased platelet counts, decreased creatinine, potassium, total protein and albumin concentrations (females).

Non-adverse changes in coagulation parameters were recorded: increased prothrombin time at 250 mg/kg/day (both sexes).

Serum levels of T4 and TSH in F0-males were decreased at 250 mg/kg/day, which was considered to be test item-related.

There were no test item-related effects on the reproductive performance.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Screening Study coornducted accding to GLP and OECD guidelines

Effects on developmental toxicity

Description of key information

The reproductive and developmental toxicity of 6-chlorohexan-1-ol, has been investigated in Wistar Han rats in a repeated dose, reproductive and developmental screening study conducted according to OECD TG 422. In the study, three respective groups of male and female rats (10 rats per group) were administered the substance daily (7 days/week) at concentrations of 25, 80 and 250 mg/kg bw/day by oral gavage administration for at least 28 days.  Control groups received a reverse osmosis water vehicle. The objectives of this study were to determine the potential toxic effects of PU-2020-905 when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 25, 80 and 250 mg/kg/day, based on   of the Dose Range Finder.

Post-implantation survival index was decreased at 25, 80 and 250 mg/kg/day., both compared to the control group of this study and to the historical control data (mean 91%, P5-P95= 83-27% (n=133). The post implantation survival index in group 4 is only lowered due to the total litter loss of female 80. At necropsy a thickened uterus was observed. Post-implantation survival index (Total Number of Offspring born as a percentage of total number of uterine implantation sites) was 94, 79, 75 and 74% for the control, 25, 80 and 250 mg/kg bw/day groups, respectively.

In Groups 2 and 4 mean litter size was significantly lower than the mean number of implantation site. However, in Group 3 the lower post-implantation survival index was caused by Female 63 with implantation site only and Female No.66 which was sacrificed during delivery.

For Female No. 48 (control), the number of pups were slightly higher than the number of implantation. This phenomenon is observed from time to time and is cased by normal sorption of these areas during lactation. No toxicological relevance was attached to this finding in the current study.

Litter size was decreased in female at 25 and 250 mg/kg/day, which was considered test item-related. Liver litter sizes were 11.6, 9.1, 11.6 and 8.6 living pups/litter for the control, 25, 80. And 250 mg/kg bw/day groups, respectively.

The concurred control range of liver litter size is 8-14 living pups/litter. There are 4/9 and 2/8 females with litter size below the lower end of the concurrent control range in Groups 2 and 4, respectively. A clear dose-relation is not apparent.

Slightly longer duration of gestation at 250 mg/kg/day may be the result of the smaller litter size in this group and is therefore likely to be an indirect effect. Considering the magnitude of change this was regarded not adverse.

Body weights of pups were decreased at 80 and 250 mg/kg/day on PND 13. Considering the magnitude of change, this finding was regarded non-adverse at 80 mg/kg/day but adverse at250 mg/kg/day.

 A trend towards lower body weights was also apparent at 80 mg/kg bw /day (7 an 6% lower than control for males and females on PND 13), respectively and not statistically significant)

No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

Screening study conducted according to GLP and OECD Test guidelines

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Justification for classification or non-classification

No effects on reproductive or developmental parameters were observed in a repeated dose and reproductive/developmental toxicity screening study conducted according to OECD TG 422. The substance does not have the potential to cause reproductive or developmental toxicity and does not require classification in respect of this endpoint according to Regulation (EC) 1272/2008.