1-Propanol, 2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 dec 2011 to 27 mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

416-210-4

EC Name:

-

Cas Number:

128119-70-0

Molecular formula:

C14H26O2

IUPAC Name:

2-methyl-3-({1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}oxy)propan-1-ol

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl:WI(Han).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany. Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight range at start of treatment was 280 - 284 gr (males) or 196 - 201 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
Lactation: Pups were kept with the dam until termination in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage-enrichment were supplied.
- Diet: Free access to prepared powder diets. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 03 feb to 27 mar 2012

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

- Method of formulation: The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the rest of the diet. The control animals and animals in the acclimatization period received similarly prepared diets but without the test substance.
- Storage conditions of formulations: In the freezer (at ≤-15ºC) until day of use. Daily portions of powder diet were defrosted and offered to the animals on a daily basis.
- Stability of powder diet: At least 8 days stable in the freezer (concentration range of 1.000 to 10.000 ppm; determined during NOTOX Project 498287). Stability at room temperature for 1 day was confirmed during this project.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or 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.
- After 14 days of unsuccessful pairing two females who had not shown evidence of mating were separated from there males.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (08 February 2012), according to a validated method (NOTOX Project 498287). Dietary samples were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability over 1 day at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

In addition, random samples were taken and stored at ≤-15°C for possible future analysis. Any remaining samples will be discarded after approval by the sponsor, or at finalization of the study report.

The accuracy of diet preparations was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Diet preparations were considered stable if the relative difference before and after storage was maximally 10%.

The concentrations analysed in the powder diets of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). No test substance was detected in the Group 1 diets. The powder diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Powder diets at the entire range were stable when stored at room temperature for at least 1 day.

Duration of treatment / exposure:

Males were exposed for 32 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 5 days of lactation.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Ad libitum.

Details on study schedule:

- Age at mating: Approximately 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

2 000 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

4 000 ppm (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 14-day dose range finding study (NOTOX Project 498284; see attached results). At ca. 561 mg/kg bw/day (7600 ppm) both female rats died in the dose range finder. Therefore, dose levels for the main study were selected to be 1.000, 2.000 and 4.000 ppm.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

- Mortality / Viability: At least twice daily.

- Clinical signs: Daily, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

- Body weights: Males and females were weighed on the first day of exposure, on Day 4 and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

- Food consumption: Daily until Day 29 of study for the males and Day 4 of lactation for the females, except for males and females which were housed together for mating and for females without evidence of mating.

- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

- General reproduction data Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

- Clinical laboratory investigations: Blood samples were collected at the end of treatment from all animals under anaesthesia using isoflurane. The animals were not fasted before blood sampling. Blood samples (0.5 mL) for haematological parameters were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA. The following haematology parameters were determined in blood prepared with EDTA as an anticoagulant, using the ADVIA® 120 Hematology System: White blood cells, Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (males of the control and high dose group).

Litter observations:

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

Postmortem examinations (parental animals):

Necropsy
- The animals were not deprived of food. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated. Necropsy was
conducted on the following days: Females: Lactation Days 5-7 and Males: Following completion of the mating period (a minimum of 28 days of dose administration). All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females.
- Samples of the following tissues and organs were collected and fixed in 10% buffered formalin: Cervix, Clitoral gland, Coagulation gland, Epididymides (Fixed in modified Davidson's solution), Kidneys, Liver, Preputial gland, Prostate gland, Seminal vesicles, Testes (Fixed in modified Davidson's solution), Uterus, Vagina, All gross lesions

Organ weights
- The following organ weights and terminal body weight were recorded from all F0-animals on the scheduled day of necropsy: Epididymides, Kidneys, Liver, Testes

Histotechnology
- All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin. From the males of the control and high dose group additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.

Histopathology:
The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4 to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- Based on possible treatment related findings: epididymides of the males of Groups 2 and 3, kidneys of both sexes of all groups, and livers of males of all groups.
A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical signs (i.e. piloerection) were noted for females at 4.000 ppm. This was seen for nine animals for 2 to 23 days during post-coitum and lactation. Piloerection was also noted for single females of the control, low and mid dose group. At this low incidence, it was not considered toxicologically significant.

Additional information on clinical signs can be found in Appendix 1 of the attached study report. (Page 28)

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first four days of treatment, body weight loss was noted for females of all dose groups in a dose related manner.
- At 1.000 ppm, this recovered to normal levels already on Day 8 of study.

- At 2.000 ppm, reduced body weights were noted during the remainder of the study however with a normal body weight gain, and this was therefore considered not toxicologically relevant.

- At 4.000 ppm, body weights for females remained slightly reduced when compared to their concurrent controls for the rest of the study.
-- A statistically significant 5.3 - 7.0% lower body weight during pre-mating and mating
-- A statistically significant 5.5 - 13.0% lower body weight during gestation
-- A statistically significant 10.8 - 12.1% lower body weight during lactation
Additionally at 4.000 ppm, a treatment related, statistically significant, reduction in body weight gain was noted during the mating (negative body weight gain), pre-mating (77% lower) and gestation (21 - 35% lower).

For males of all dose levels, body weights and body weight gain remained in the same range as controls over the treatment period.

Additional information on bodyweight and body weight gain can be found in Appendix 1 of the attached study report (Page 29 to 34).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 4.000 ppm, food consumption was moderately decreased during the first day of treatment for the males (14 gram versus 19 gram) and severely reduced during the first two days of treatment for the females (5-6 gram versus 14 gram).

At 2.000 ppm, females showed a moderately reduced food consumption on Days 1 and 2 (10-11 gram versus 14 gram).

In addition, during post-coitum (mean 23% lower) and lactation (mean 24% lower) reduced food consumption was noted at 4.000 ppm. This was statistically significant from Day 3 post-coitum onwards.

All other statistically significant changes were within normal limits, and were therefore not considered toxicologically significant.

Food consumption before or after allowance for body weight was similar between animals treated at 1.000 ppm (both sexes) and 2.000 ppm (males) when compared to the control animals.

Additional information on food consumption can be found in Appendix 1 of the attached study report (Page 35 to 40).

Haematological findings:

no effects observed

Description (incidence and severity):

For haematology parameters, no treatment related findings were noted up to 4.000 ppm for both sexes.

Additional information on haematology can be found in Appendix 1 of the attached study report (Page 44 to 45).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were treatment related morphologic alterations in the male liver and kidneys.
Liver:
• Hepatocellular hypertrophy was noted at minimal degree in 3/10 males at 4.000 ppm.

Kidneys:
• A dose related increase in incidence and severity of hyaline droplets from 0/10 in the control group, 7/10 (minimal-slight) in the 1000 ppm male rats, 10/10 (minimal-moderate) in the 2000 ppm male rats to 10/10 (slight-moderate) in the 4000 ppm male rats.
• Increased incidence (9/10) and severity (up to slight) of tubular basophilia in the 4000 ppm male rats compared to minimal in 4/10 of the control group, 5/10 of the 1000 ppm male rats and 4/10 of the 2000 ppm male rats.
• Increased incidence (4/10) and severity (up to slight) of tubular necrosis and/or granular cast(s) in the 4000 ppm male rats compared to 0/10 in the control group, 1/10 (minimal) in the 1000 ppm male rats and 0/10 in the 2000 ppm male rats.

There were two microscopic findings of note in the kidneys. See macroscopic examination.

Additional information on histopathology can be found in Appendix 4 of the attached study report (Page 172 - 239).

Reproductive function / performance (P0)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all Group 1 and all Group 4 males evaluated

Reproductive performance:

no effects observed

Description (incidence and severity):

- There were no treatment-related findings in the examined ovaries, epididymides and testes.
- Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
- Gestation index and duration of gestation were within normal limits for all groups.
- Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Additional information on reproduction can be found in Appendix 1 of the attached study report (Page 51 - 53).

Details on results (P0)

Analysis of dose preparations:
- The concentrations analysed in the powder diets of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). No test substance was detected in the Group 1 diets. The powder diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Powder diets at the entire range were stable when stored at room temperature for at least 1 day.

Test material intake for 1000, 2000, and 4000 ppm, respectively
- Males, premating: 70, 139, 283 mg/kg bw
- Males, mating: 64, 124, 252 mg/kg bw
- Females, premating: 73, 144, 264 mg/kg bw
- Females, post-coitum: 85, 168, 299 mg/kg bw
- Females, lactation: 106, 238, 395 mg/kg bw

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

3

Key result

Dose descriptor:

NOAEL

Remarks:

Fertility

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: body weight and food consumption

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Incidental findings consisted of scabs (on the hindleg or flank) and a wound (on the hindleg). The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Additional information on pup data can be found in Appendix 1 of the attached study report (Page 55).

Mortality / viability:

no mortality observed

Description (incidence and severity):

Two pups of the control group and one pup at 1.000 ppm were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. 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.

Additional information on pup data can be found in Appendix 1 of the attached study report (Page 55).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight of male and female pups at 4.000 ppm was lower than controls on Days 1 (13%) and 4 (16%), achieving a level of statistical significance. Weight gain over Days 1-4 was considered normal (47% for Group 4 compared to 51% for Group 1). The reduced body weights of the pups at 4.000 ppm was considered a secondary effect caused by the reduced maternal body weights as body weight gain of the pups over Days 1 to 4 of lactation was unaffected.

Additional information on pup body weight can be found in Appendix 1 of the attached study report (Page 55).

Gross pathological findings:

no effects observed

Description (incidence and severity):

Absence of milk in the stomach was noted for one pup that was found dead. Incidental findings for surviving pups consisted of a scab on the abdomen or hindleg, and a wound on the cheek. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Additional information on pup data can be found in Appendix 2 of the attached study report (Page 55).

Other effects:

no effects observed

Description (incidence and severity):

Early postnatal pup development: Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, and external macroscopy did not reveal treatment-related findings.

Additional information on pup data can be found in Appendix 1 of the attached study report (Page 55).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD 421, GLP), the systemic NOAEL was determined 124 and 144 mg/kg bw for male and females, respectively. The reproduction NOAEL was at least 252 mg/kg bw. The developmental NOAEL was 168 mg/kg bw, but considered a secondary effect.

Executive summary:

A dietary reproscreen study according to OECD TG 421 was performed following GLP. The dosing was nominally 1000, 2000 and 4000 ppm in the diet to result in 250, 125 and 60 mg/kg bw. Ten males and ten females were exposed per dose level. Males were exposed for 32 days, i.e. 2 weeks prior to mating, during mating, and until termination. Females were exposed for 42-54 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 5 days of lactation. The following repeated dose toxicity parameters were recorded: mortality, clinical signs body weight and food consumption, haematology parameters and organ weights of liver, kidney, testis and epididymides. These organs were also microscopically evaluated. For fertility the following parameters were evaluated: mating, fertility, conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturation and maternal care. For developmental toxicity the following parameters are evaluated: sex ratio, early postnatal pup development including mortality, clinical signs, body weight and macroscopy. Accuracy, homogeneity and stability of the powder diets were demonstrated by analyses.

Results: Dietary analysis showed that the concentrations were within 80 -120% of the nominal. The intake at the highest dose resulted in an intake of 252 -283 mg/kg bw for males and 264 -395 mg/kg bw for females. At the mid dose these results were 124 -139 mg/kg bw and 144 -238 mg/kg bw, respectively. At the low dose these results were 64 - 70 mg/kg bw and 73 -106 mg/kg bw, respectively.

Parental results: Mortality: No mortality occurred during the study period. Clinical signs: Toxicity consisted of the occurrence of piloerection at 4000 ppm. Body weight: In females, at 4000 ppm, body weights was statistically significant reduced during the entire study: 5.3 - 7.0% lower body weight during pre-mating and mating, 5.5 - 13.0% lower body weight during gestation, and 10.8 - 12.1% lower body weight during lactation. A treatment related, statistically significant, reduction in body weight gain was noted during the mating (negative body weight gain), pre-mating (77% lower) and gestation (21 - 35% lower). For males of all dose levels, body weights and body weight gain remained in the same range as controls over the treatment period. Food consumption: In females, at 4000 ppm, post-coitum (mean 23% lower) and lactation (mean 24% lower) food consumption was reduced. Haematology parameters: Similar for all groups male and female. Organ weight: Liver weights were increased for males treated at 2.000 and 4.000 ppm: Absolute 5 and 15% and Relative: 9 and 16%, respectively. Macroscopy: Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. Microscopy: Hepatocellular hypertrophy was noted at minimal degree in 3/10 males at 4.000 ppm. Microscopic findings in the kidneys consisted of a dose related increase in incidence and severity of hyaline droplets in all treatment groups. Hyaline droplets were considered to represent alpha2μglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. Therefore the presence of hyaline droplets was considered not to be adverse at 1.000 and 2.000 ppm. However, at 4.000 ppm there were associated findings consisting of tubular necrosis, granular cast(s) and tubular basophilia. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium which may then result in tubular cell degeneration. These changes are exclusively found in male rats. Therefore, these findings are not predictive for man and their occurrence has no relevance in human safety assessment. 

Fertility: No reproduction toxicity was observed up to 4.000 ppm.

Developmental: No direct developmental toxicity was observed up to the highest concentration tested (4.000 ppm). The only effect observed was reduced body weight of male and female pups at 4.000 ppm was on Days 1 (13%) and 4 (16%), achieving a level of statistical significance. Weight gain over Days 1-4 was considered normal (47% for Group 4 compared to 51% for Group 1). Therefore, the reduced body weights of the pups at 4.000 ppm was considered a secondary effect caused by the reduced maternal body weights as body weight gain of the pups over Days 1 to 4 of lactation was unaffected.

Conclusion: Treatment with Bornafix by dietary administration in male and female Wistar Han rats at dose levels of 1.000, 2.000 and 4.000 ppm revealed parental toxicity at 4.000 ppm for females and males. No reproduction toxicity was observed for treatment up to 4.000 ppm. Developmental toxicity at 4.000 ppm consisted of reduced pup body weights, which was considered a secondary effect. A parental NOAEL of 2.000 ppm was established for males and females (124 and 144 mg/kg bw, respectively). The reproduction NOAEL was at least 4.000 ppm (>252 mg/kg bw) and the developmental NOAEL was 2.000 ppm (based on a secondary effect) (168 mg/kg bw).