6,6-dimethoxy-2,5,5-trimethylhex-2-ene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

OECD 422 study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

6,6-dimethoxy-2,5,5-trimethylhex-2-ene

EC Number:

266-885-2

EC Name:

6,6-dimethoxy-2,5,5-trimethylhex-2-ene

Cas Number:

67674-46-8

Molecular formula:

C11H22O2

IUPAC Name:

6,6-dimethoxy-2,5,5-trimethylhex-2-ene

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Unknown

IUPAC Name:

Unknown impurities

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Test Item 205601/B
Identification Methyl Pamplemousse
Appearance Colourless to pale yellow liquid
Batch PE00170602
Purity/Composition See Certificate of Analysis
Test item storage At room temperature protected from light
Stable under storage conditions until: 11 March 2017 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The accuracy of diet preparations is considered acceptable if the mean measured
concentrations are 80-120% of the target concentration. Homogeneity is demonstrated if the
coefficient of variation is ≤ 10%.
Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500
to 15000 ppm was confirmed for at least 3 weeks in the freezer (≤-15°C) and for 1 day at
room temperature (Test Facility Study No.507072 (method development and validation
study)).
In addition, random back-up diet samples will be taken from diets used for analytical
sampling and stored at ≤-15ºC for possible future analysis. Any remaining samples at
finalization of the study report will be discarded.
If the analytical determinations are not performed on the day of preparation, the samples will
be stored at ≤-15ºC.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat: Crl:WI(Han) (outbred, SPF-Quality). Untreated, nulliparous, non-pregnant females and untreated males will be used at the initiation of the study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
48 females and 40 males.
Acclimatisation: At least 5 days prior to start of pretest (females) or treatment (males).
Environmental controls for the animal room are set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle may be interrupted for study related activities. Any variations to these conditions will be evaluated and maintained in the raw data.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.
The actual test item intake will be estimated based on the body weight and food consumption values.

Details on mating procedure:

Pretest Females will be housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating Animals will be housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating Males and females will be cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating Males will be housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females will be individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses will be conducted on a single occasion during the treatment phase on samples as specified below, according to a validated method (Test Facility Study No. 507072):
The accuracy of diet preparations is considered acceptable if the mean measured concentrations are 80-120% of the target concentration. Homogeneity is demonstrated if the coefficient of variation is ≤ 10%.
Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500 to 15000 ppm was confirmed for at least 3 weeks in the freezer (≤-15°C) and for 1 day at
room temperature (Test Facility Study No.507072 (method development and validation study)).
In addition, random back-up diet samples will be taken from diets used for analytical sampling and stored at ≤-15ºC for possible future analysis. Any remaining samples at finalization of the study report will be discarded.
If the analytical determinations are not performed on the day of preparation, the samples will be stored at ≤-15ºC.

Duration of treatment / exposure:

The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.

Frequency of treatment:

The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.

Details on study schedule:

Source F0 Charles River Laboratories France, L'Arbresle Cedex, France or Charles River Deutschland, Sulzfeld, Germany. Details will be documented in raw data and report.
Age at start pretest Females: approximately 10-12 weeks.
Age at start F0-treatment Males: approximately 10-12 weeks.
Females: approximately 12-14 weeks.
Number of F0-animals 48 females and 40 males.
A total of 40 females will be selected at randomization before initiation of the pretest phase. Any selected female without at least two regular estrous cycles at the end of the pretest phase will be replaced by one of the 8 additional females having at least two regular estrous cycles. A total of 40 females with at least two regular estrous cycles will continue in the study. The supernumerary females will then be removed from the study,
and their estrous cycle results will be kept in the raw data but will not be reported.
Acclimatization F0 At least 5 days prior to start of pretest (females) or treatment (males).
Health inspection F0 At least upon receipt of the animals.
Randomization F0 Before initiation of pretest, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0 During pretest (females1) and treatment (males and females): by earmark and tattoo.
Mating procedures Following a minimum of 14 days of treatment for the males and females, they will be cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating will be confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day will be designated Day 0 post-coitum. Once mating has occurred, the males and females will be separated.
A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male of proven fertility of the same group for a maximum of 7 days (if possible).
Parturition The females will be allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Females that are littering will be left undisturbed.
Number of pups Approximately 480 pups (40 litters x 12 pups).
Identification of pups On PND 1, all pups will be randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
When general hair growth blurs the identification, the pups will be identified by tattoo on the feet.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm

Remarks:

1500 ppm in the diet corresponded to mean daily test item intake levels of 113 mg/kg bw/day in males, and 204 mg/kg bw/day in females.

Doses / concentrations 2

Dose / conc.:

5 000 ppm

Remarks:

5000 ppm in the diet corresponded to mean daily test item intake levels of 386 mg/kg bw/day in males, and 615 mg/kg bw/day in females.

Doses / concentrations 3

Dose / conc.:

15 000 ppm

Remarks:

15,000 ppm in the diet corresponded to mean daily test item intake levels of 1102 mg/kg bw/day in males, and 1458 mg/kg bw/day in females.

No. of animals per sex per dose:

48 females and 40 males.

Control animals:

yes, concurrent no treatment

Details on study design:

Method Oral, by inclusion in the diet.
Frequency Ad libitum.
Dietary Inclusion Levels The amount of test item incorporated into the diet will be kept at a constant level in terms of ppm, throughout the study period.
The actual test item intake will be estimated based on the body weight and food consumption values.
Exposure period Males will receive test diet for a minimum of 28 days, up to and including the day before scheduled necropsy. This includes a minimum of two weeks prior to mating and during the mating period.
Females will receive test diet for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least thirteen days after delivery, up to and including the day before scheduled necropsy.

Positive control:

NA

Examinations

Parental animals: Observations and examinations:

Mortality / Viability At least twice daily. Animals showing pain, distress or discomfort, which is considered not transient in nature or is likely to become more severe, will be sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death will be recorded as precisely as possible.
Clinical signs Once prior to first administration and at least once daily from start of treatment onwards, detailed clinical observations will be made for all animals.

Body weights Males and females will be weighed on the first day of exposure and weekly thereafter. Mated females will be weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. In order to monitor the health status animals may be weighed more often. This will be documented in the study raw data.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Weekly, except for males and females which are housed together for mating and for females without evidence of mating.
Food consumption of mated females will be 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 Subjective appraisal will be maintained during the study period
and a quantitative assessment introduced in the event of a suspected treatment related effect.

Oestrous cyclicity (parental animals):

Estrous cycle Daily vaginal lavage will be performed to determine the stage determination of estrus beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of
copulation is observed. Vaginal lavage will continue for those females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage will be taken to determine the stage of estrus.

Sperm parameters (parental animals):

No

Litter observations:

Each litter will be examined to determine the following, if practically possible:
Mortality / Viability The numbers of live and dead pups will be determined on PND 1 and daily thereafter. If possible, defects or cause of death will be evaluated. Animals showing pain, distress or discomfort, which is considered not transient in nature or is likely to become more severe, will be sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
Clinical signs At least once daily, detailed clinical observations will be made for all animals. Only days on which clinical signs are present between first and last litter check will be given in the respective report tables.
Body weights Live pups will be weighed on PND 1, 4, 7 and 13.
Sex Sex will be determined for all pups on PND 1 and 4.
Anogenital distance Anogenital distance (AGD) will be measured for all live pups on PND 1. The AGD will be normalized to the cube root of body weight.
Areola/nipple retention On PND 13, all males in each litter will be examined for the number of areola/nipples.

Postmortem examinations (parental animals):

All animals will be deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water will be available.
All animals surviving to the end of the observation period and all moribund animals will be deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities will be recorded.

Necropsy will be conducted on the following days:
Condition Day of necropsy
Males Following completion of the mating period (a minimum of 28 days of dose administration).
Females which deliver PND 14-16.
Females which fail to deliver Post-coitum Days 25-27 (females with evidence of mating) or approximately 24-26 days after the last day of the mating period (females without evidence of mating).
Females with total litter loss Within 24 hours of litter loss.
Spontaneous deaths4 As soon as possible after death and always within 24 hours.
Euthanized in extremis6 When pain, distress or discomfort is considered not transient in nature or is likely to become more severe.
The numbers of former implantation sites will be recorded for all paired females. In case no macroscopically visible implantation sites are present, nongravid uteri will be stained using the Salewski technique (Ref. 1) in order to detect any former implantation sites (Salewski staining prepared at Charles River Den Bosch using Ammoniumsulfide-solution 20% (Merck,
Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
For females which fail to deliver a complete nest, uterine contents (i.e. any fetuses, placenta and implantation sites) will be fixed (if applicable), but will not be examined histopathologically in first instance.
Samples of the tissues and organs specified on the next page will be collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Postmortem examinations (offspring):

Pups, younger than 7 days will be euthanized by decapitation.
All remaining pups (PND 7-15) will be sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater, The Netherlands) by intraperitoneal (ip) injection.
Pups found dead during the weekend will be fixed in identified containers containing 70% ethanol (Klinipath, Duiven, The Netherlands) if not necropsied on the same day.
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) will be collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter will be collected into one serum tube. For further details, see section 3.10: Blood sampling for thyroid hormone analysis.
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples will be collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) will be drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination. Blood will be collected into serum tubes.

Statistics:

The following statistical methods will be used to analyse the data:
• If the variables can be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one t-test) based on a pooled variance estimate will be applied for the
comparison of the treated groups and the control groups for each sex.
• The Steel-test (Ref. 3; many-to-one rank test) will be applied if the data cannot be assumed to follow a normal distribution.
• The Fisher Exact-test (Ref. 4) will be applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) will be applied to motor activity data to determine intergroup differences. In case intergroup differences are seen, the Wilcoxon test (Ref. 6) will be applied to compare the treated groups to the control group.
All tests will be two-sided and in all cases p < 0.05 will be accepted as the lowest level of significance. Group means will be calculated for continuous data and medians will be calculated for discrete data (scores) in the summary tables. Test statistics will be calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
Clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 15,000 ppm, body weight gain and body weights of males were statistically significantly lower from Day 1 of the mating period. Mean body weights were 8% lower compared to controls at the end of treatment. Females at 15,000 ppm also showed slightly lower body weight gain being statistically significantly at Days 7-14 of the post-coitum period.
Thereafter, their weight gain tended to return to control values. Mean body weights were less than 5% lower compared to controls.
It should be remarked that the control group means at post-coitum Days 17 and 20 were somewhat depressed by the lower weight gain of two females (nos. 44 and 47) which had only one implantation site and delivered no offspring. Thus, the difference in weight gain between 15,000 ppm females delivering offspring and controls was somewhat larger than indicated by the group means in the summary table.
Body weight and body weight gain of 15,000 ppm females during the pre-mating and lactation periods were considered not to be affected by treatment.
Other statistically significant variations in body weight gain at 1500 and 5000 ppm occurred in the absence of a dose-related response, and were therefore considered to be unrelated to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 15,000 ppm, food consumption before and after correction for body weight was reduced for both sexes over Days 1-2 of the premating period and throughout the post-coitum and lactation period, achieving a level of statistical significance on most occasions. Mean of mean absolute food consumption was approximately 31% and 40% lower than control mean for the post-coitum and lactation phase, respectively. At 5000 ppm, food consumption of females before and after correction for body weight also lower during post-coitum and lactation; a level of statistical significance was only achieved on a few occasions, and mean of mean absolute food consumption was approximately 20% and 18% lower than control mean for the post-coitum and lactation phase, respectively.
For male groups, variations in food intake were recorded across the dose groups that showed no clear dose-related response. Also, mean over mean food consumption showed no clear relationship to the administered dose.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The mean daily intake of the test item per kg body weight during the different phases of the study is presented in the study report, section 7.2.6.

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

At 15,000 ppm, statistically significantly higher platelet counts and delayed prothrombin time (PT) were recorded for females. Means remained within the range considered normal for rats of this age and strain7.
The statistically significantly reduced prothrombin time in males at 5000 and 15,000 ppm was not considered to be related to treatment due to the lack of a dose-related response.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 15,000 ppm, lower glucose and higher chloride were recorded for females. Means remained within the range considered normal for rats of this age and strain7. For glucose, the control mean was relatively high At 1500 and 5000 ppm, clinical biochemistry parameters were similar to control means.
Thyroid hormone analyses: Serum levels of T4 in F0 males were not considered to be affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

A lower hind limb grip strength was recorded at 15,000 ppm in males, which was below the range normally encountered for rats of this age and strain. However, there were no corroborative clinical signs or changes in other measures in the neuromuscular domain (including forelimb grip strength, gait, air righting reflex and motor activity). Therefore, this difference in hind limb grip strength was not considered to reflect an adversely impaired neuromuscular function. There were no treatment-related changes in other in-life parameters including mortality and clinical appearance and other functional observation parameters up to 15,000 ppm.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment.
Most females had regular cycles, generally of 4 days. Extended di-estrus during pairing occurred in one control female (no. 41) and two treated females had irregular cycles during the pre-mating period (nos. 59 and 76 at 1500 and 15,000 ppm, respectively). These females had normal litters. These cycle abnormalities were considered unrelated to treatment due to their incidental occurrence and lack of a dose-related trend.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Mating index was not affected by treatment. All females showed evidence of mating.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Number of implantation sites was considered to be unaffected by treatment.
Two control females (nos. 44 and 47; both had no offspring) each had a single implantation site only.
At 5000 and 15,000 ppm, fertility index was reduced, i.e. 70% vs 100 and 90% at 0 and 1500 ppm, respectively. The number of pregnant females at 5000 and 15,000 ppm (7/10) was lower than normally seen in untreated rats.
The following treated females were not pregnant despite evidence of mating: no. 58 of Group 2, nos. 61, 62 and 70 of Group 3, and nos. 74, 78 and 80 of Group 4.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

5000 ppm

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

food consumption and compound intake

Target system / organ toxicity (P0)

Critical effects observed:

no

Lowest effective dose / conc.:

5 000 ppm

Organ:

other: Food intake

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 98, 99, 94 and 98% at 0, 1500, 5000 and 15,000 ppm, respectively.
Two pups of the control group (litter no. 46), one pup at 1500 ppm (litter no. 59), four pups at 5000 ppm (litter no. 64), and one pup at 15,000 ppm (litter no. 77) went missing (presumably cannibalized) at PND 2-4. This post-natal loss was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 15,000 ppm, mean body weights of male and female pups were reduced at PND 7 and 13 (statistically significant on PND 13 for both sexes). Relative differences from controls were approximately 15 and 30% on PND 7 and 13, respectively).
Pup body weights at the lower dose levels (1500 and 5000 ppm) were not considered affected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: All of the above measures

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

15 000 ppm

System:

other: All tissues

Organ:

other: All tissues

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

5 000 ppm

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

Parental NOAEL: 1500 ppm1, based on renal changes in males at 5000 ppm (combination of hyaline droplet accumulation, tubular basophilia and granular casts, and associated higher renal weight). Given that the renal changes are considered to represent a male rat specific response, a NOAEL of 5000 ppm3 may be considered for risk assessment purposes based on the significantly lower food intake of females at 15,000 ppm.
Reproduction NOAEL: at least 15,000 ppm2.

Executive summary:

NOAEL for risk assessment is 5000 ppm (615 mg/kg bw/day) based on reduced food consumption of females.