Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is no evidence indicating that isophorone interferes adversely with the reproduction. No changes were observed in pregnancy rates, litter sizes and pups abnormalities (Dutertre-Catella, 1976) or in histopathological examinations of the reproduction organs after long-term studies (NTP, 1986; Rohm Haas Co., 1972; see chapter 7.8.3 of IUCLID 5). A data waiver is claimed.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a limited one generation study, 10 male and 10 female Wistar rats were exposed to 2873 mg/m3 (500 ppm) isophorone in air (Dutertre-Catella, 1976). Treatment with isophorone did not influence pregnancy rates and litter sizes nor were there any abnormalities observed in the pups. However only one isophorone concentration was used, the group size was small, and no information was provided on reproductive success and maternal survival. Under the conditions on this study, it can be concluded that isophorone has no adverse effect on reproduction of rats.

The histological examination of the reproductive organs of male and female mice and rats (mammary gland, seminal vesicle, prostate/testis or ovary/uterus) treated orally with up to 1000 mg isophorone/kg for 13 weeks did not reveal any adverse effects after macroscopic and microscopic examination (NTP, 1986; see chapter 7.8.3 of IUCLID 5).

In a 90 days study with male and female beagle dogs (4 animals/dose/sex were administered up to 150 mg isophorone/kg/day) no changes were reported either after histopathological examination of testes, prostate, seminal vesicles and ovary, uterus, mammary gland, respectively (Rohm & Haas Co., 1972; see chapter 7.8.3 of IUCLID 5).



Justification for selection of Effect on fertility via oral route:
According to section 1.2 of Annex XI, the study need not be done if there is a weight of evidence to conclude the substance does not have a particular property, and further testing on vertebrate animals may be omitted. Furthermore Annex X states :"When, for certain endpoints, it is proposed not to provide information for other reasons than those mentioned in column 2 of this Annex or in Annex XI, this fact and the reasons shall also be clearly stated."
Justification for waiving: For isophorone a one-generation study was conducted. Treatment with isophorone did not influence pregnancy rates and litter sizes nor were there any abnormalities in the pups (Dutertre-Catella, 1976, chapter 7.8.1 of IUCLID 5). In an inhalation teratogenicity study with rats and mice, isophorone was neither embryotoxic nor teratogenic up to the highest concentration tested in the study (Exxon, 1984, see chapter 7.8.2 of IUCLID 5). Furthermore there are no indications of adverse effects on the examined reproductive organs of rats, mice and dogs in several 90 day studies (NTP, 1986 and Rohm & Haas, 1972, see chapter 7.8.3 of IUCLID 5). All this findings lead to the conclusion that effects on fertility of the substance isophorone at doses, which do not cause parental toxicity, are unlikely. Therefore further studies regarding effects on fertility are not necessary for isophorone.

Justification for selection of Effect on fertility via inhalation route:
According to section 1.2 of Annex XI, the study need not be done if there is a weight of evidence to conclude the substance does not have a particular property, and further testing on vertebrate animals may be omitted. Furthermore Annex X states :"When, for certain endpoints, it is proposed not to provide information for other reasons than those mentioned in column 2 of this Annex or in Annex XI, this fact and the reasons shall also be clearly stated."
Justification for waiving: For isophorone a one-generation study was conducted. Treatment with isophorone did not influence pregnancy rates and litter sizes nor were there any abnormalities in the pups (Dutertre-Catella, 1976, chapter 7.8.1 of IUCLID 5). In an inhalation teratogenicity study with rats and mice, isophorone was neither embryotoxic nor teratogenic up to the highest concentration tested in the study (Exxon, 1984, see chapter 7.8.2 of IUCLID 5). Furthermore there are no indications of adverse effects on the examined reproductive organs of rats, mice and dogs in several 90 day studies (NTP, 1986 and Rohm & Haas, 1972, see chapter 7.8.3 of IUCLID 5). All this findings lead to the conclusion that effects on fertility of the substance isophorone at doses, which do not cause parental toxicity, are unlikely. Therefore further studies regarding effects on fertility are not necessary for isophorone.

Justification for selection of Effect on fertility via dermal route:
According to section 1.2 of Annex XI, the study need not be done if there is a weight of evidence to conclude the substance does not have a particular property, and further testing on vertebrate animals may be omitted. Furthermore Annex X states :"When, for certain endpoints, it is proposed not to provide information for other reasons than those mentioned in column 2 of this Annex or in Annex XI, this fact and the reasons shall also be clearly stated."
Justification for waiving: For isophorone a one-generation study was conducted. Treatment with isophorone did not influence pregnancy rates and litter sizes nor were there any abnormalities in the pups (Dutertre-Catella, 1976, chapter 7.8.1 of IUCLID 5). In an inhalation teratogenicity study with rats and mice, isophorone was neither embryotoxic nor teratogenic up to the highest concentration tested in the study (Exxon, 1984, see chapter 7.8.2 of IUCLID 5). Furthermore there are no indications of adverse effects on the examined reproductive organs of rats, mice and dogs in several 90 day studies (NTP, 1986 and Rohm & Haas, 1972, see chapter 7.8.3 of IUCLID 5). All this findings lead to the conclusion that effects on fertility of the substance isophorone at doses, which do not cause parental toxicity, are unlikely. Therefore further studies regarding effects on fertility are not necessary for isophorone.

Effects on developmental toxicity

Description of key information
In two guideline comparable inhalation teratogenicity studies with rats and mice, the NOAECs for maternal toxicity were 289 mg/m3 (based on < 7 % reductions in body weight gains) (Exxon, 1984). Isophorone was neither embryotoxic nor teratogenic up to the highest test concentration of 664 mg/m3 isophorone. Results of the present DRF study with F344 rats (Vivotecnia, 2016) together with the information existing in the published literature, provide clear evidence that F344 rats are not a suitable strain for prenatal developmental toxicity studies. Furthermore, based on the results of this study with Wistar rats, the NOAEL for maternal toxicity and embryo-fetal development was 150 ppm (Vivotecnia, 2016).
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-12-21 to 1984-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
Method: guideline comparable inhalation teratogenicity test
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
TEST ORGANISMS
- Source: Harlan Sprague Dawley Inc.
- Age at study initiation: 11 weeks
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days
- Housing: animals were contained in a 12.9 m3 stainless steel and glass chamber and exposed either to conditioned air or to an atmosphere of
isophorone suspended in air
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
ADMINISTRATION / EXPOSURE
- Vehicle: no vehicle
- Concentrations: 0 / 25 / 50 / 115 ppm (corresponds to 0, 144, 289 and 664 mg/m3)
- Type or preparation of particles: vapor
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All chambers were routinelly sampled on approximately an hourly basis by gas chromatograpgy.
Study mean concentrations as determined by GC were: group I = 0 ppm, group II = 27 ppm, group III = 52 ppm, group IV = 111 ppm

Details on mating procedure:
MATING PROCEDURES: 
Virgin female Fischer rats (approximately 11 weeks of  age) were paired with similar males for mating. Females were confirmed to  have mated by 
observation of a copulatory plug in the vagina or by  observation of sperm in a vaginal rinse. Confirmed mated females were assigned to groups
randomly.
Duration of treatment / exposure:
6th to 15th day of gestation
Frequency of treatment:
6 h/d
Duration of test:
Rats were sacrified by methoxyflurane inhalation and examined by gross necropsy on the 20th day of gestation.
No. of animals per sex per dose:
22 female rats
Control animals:
other: yes, concurrent conditioned air
Details on study design:
Sex: female
Duration of test: section on the 20th d of gestation
Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Body weight gain: each 3rd day
- Food consumption: 3 day intervals
- Clinical observations: each 3rd day

Ovaries and uterine content:
- Examination of uterine content: identified as live fetuses, dead  fetuses, late resorptions, and early resorptions at end of study
(day 20  of gestation). The uterus of each animal was stained in 10 % aqueous  ammonium sulfide and further examined for confirmation of 
implantation  sites. Corpora lutea were counted.
Fetal examinations:
Examination of fetuses: Live and dead fetuses were weighed, examined  externally for gross abnormalities, and crown-rump distances were  
determined. One half of the fetuses from each litter were decapitated, the heads were preserved and subsequently sectioned and examined, viscera
of these fetuses were examined by the Staples technique. All fetuses were eviscerated and processed for skeletal staining . Only those fetuses which
had not been decapitated were examined for skeletal malformations and ossification  variations.
Statistics:
STATISTICAL METHODS: 
- Bartlett's test of homogeneity of variance: body weight, body weight  change, food consumption, number of implantation sites,  ratio of live 
fetuses to implantation sites, ratios of resorptions to implant sites,  malformations per litter.
- Kruskal-Wallis test if variances were not equivalent.
- Standard nested analysis of variance for fetal weights.
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality and day of death: no mortalities
- Number pregnant per dose level: 22
- Number of resorptions: no statistically significant differences between  treated and control groups
- Number of implantations: no statistically significant differences  between treated and control groups
- Number of corpora lutea: no statistically significant differences  between treated and control groups
- Duration of Pregnancy: no statistically significant differences between  treated and control groups
- Body weight: reduced in days 12 (-6.1 %) and 15 (-6.8 %) rats in 664  mg/m3 dose group
- Food/water consumption: reduced food consumption in 664 mg/m3 dose group
- Clinical signs: alopecia and cervical or anogenital staining (each  dose-related).
Dose descriptor:
NOAEC
Effect level:
289 mg/m³ air
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL DATA: 
No statistically significant differences between treated and control  groups:
- Litter size and weights
- Number viable
- Sex ratio
- Grossly visible abnormalities
- External abnormalities
- Soft tissue abnormalities
- Skeletal abnormalities
Dose descriptor:
NOAEC
Effect level:
>= 664 mg/m³ air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

NOAEL (maternal): based on reduced body weight, clinical signs are not considered

Conclusions:
In this inhalation teratogenicity study with rats, isophorone elicited a clinical effect in the pregnant dams in the form of decreased food consumption
(664 mg/m3, days 6-20 and 0-20), lower body weights (664 mg/m3, days 12 and 15 of gestation), and dose related increases in alopecia and
staining of the cervical and anogenital areas.
During the conduct of the probe study there was one instance of exencephaly noted in a rat fetus. Based on the observations made in this study the
authors do not believe that this anomaly was related to the test material.
Within the framework of the dose levels and test methods used, it is concluded, that isophorne was not teratogenic or fetotoxic in rats.
Executive summary:

Pregnant Fischer 344 rats were treated daily for 6 hours by inhalation (whole body) with isophorone from day 6 to day 15 of gestation to examine potential developmental toxicity effects of the test substance. Dosages used were 0 (conditioned air control), 144, 289 and 664 mg/m3, respectively (22 animals per dose level).

There was a significant reduction in food consumption of rats of the highest dose group. Body weight was also reduced in animals of the highest dose group (gestation day 12: -6.1%; gestation day 15: -6.8%). Additionally, a dose related increase in alopecia was observed, as well as a discoloration of the cervical and aogenital region. Adverse effects on the fetuses were not observed.

Therefore, under the conditions of this inhalation teratogenicity study with rats, the NOAEC for maternal toxicity was determined to be 289 mg/m3 (based on < 7% reduction in body weight gain). Isophorone was neither embryotoxic nor teratogenic up to the highest test concentration of 664 mg/m3 isophorone.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-12-21 to 1984-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
Teratogenicity Test
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
TEST ORGANISMS
- Source: Charles River Labs (Kingston Facility)
- Age at study initiation: 9 weeks
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days
- Housing: animals were contained in a 12.9 m3 stainless steel and glass chamber and exposed either to conditioned air or to an atmosphere of
isophorone suspended in air
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
ADMINISTRATION / EXPOSURE
- Vehicle: no vehicle
- Concentrations: 0 / 25 / 50 / 115 ppm (corresponds to 0, 144, 289 and 664 mg/m3)
- Type or preparation of particles: vapour
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All chambers were routinelly sampled on approximately an hourly basis by gas chromatograpgy.
Study mean concentrations as determined by GC were: group I = 0 ppm, group II = 27 ppm, group III = 52 ppm, group IV = 111 ppm
Details on mating procedure:
MATING PROCEDURES
Virgin female CD-1 mice (approximately 9 weeks of age)  were paired with similar males for mating. Females were confirmed to have mated by
observation of a copulatory plug in the vagina or by observation  of sperm in a vaginal rinse. Confirmed mated females were assigned to groups
randomly.
Duration of treatment / exposure:
6th to 15th day of gestation
Frequency of treatment:
6 h/d
Duration of test:
Mice were sacrified by methoxyflurane inhalation and examined by gross necropsy on the 18th day of gestation.
No. of animals per sex per dose:
22 female mice
Control animals:
other: yes, concurrent conditioned air
Details on study design:
Sex: female
Duration of test: section on the 18th d of gestation
Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Body weight gain: each 3rd day
- Food consumption: not determined
- Clinical observations: each 3rd day

Ovaries and uterine content:
- Examination of uterine content: identified as live fetuses, dead  fetuses, late resorptions, and early resorptions at end of study (day 18  of gestation).
  The uterus of each animal was stained in 10 % aqueous ammonium sulfide and further examined for confirmation of implantation  sites. 
Corporalutea were counted.
Fetal examinations:
- Examination of fetuses: Live and dead fetuses were weighed, examined  externally for gross abnormalities, and crown-rump distances were  
determined.  One half of the fetuses from each litter were decapitated, the heads were preserved and subsequently sectioned and examined, viscera of these fetuses were examined by the Staples technique. All fetuses were eviscerated and processed for skeletal staining . Only those fetuses which
had not been decapitated were examined for skeletal malformations and ossification  variations.

Statistics:
STATISTICAL METHODS: 
- Bartlett's test of homogeneity of variance: body weight, body weight  change, number of implantation sites, ratio of live fetuses to  implantation 
sites, ratios of resorptions to implant sites, malformations  per litter.
- Kruskal-Wallis test if variances were not equivalent.
- Standard nested analysis of variance for fetal weights.
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality and day of death: no mortalities
- Number pregnant per dose level: 22
- Number of resorptions: no statistically significant differences between  treated and control
- Number of implantations: no statistically significant differences  between treated and control
- Number of corpora lutea: no statistically significant differences  between treated and control
- Duration of Pregnancy: not statistically significant differences  between treated and control
- Body weight: reduced in day 18 mice in 664 mg/m3 dose group (-5.6 %,  corrected for uterine weight)
- Clinical signs: Unremarkable
Dose descriptor:
NOAEL
Effect level:
289 mg/m³ air
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL DATA: 
- Litter size and weights: no statistically significant differences  between treated and control
- Number viable: no statistically significant differences between treated  and control
- Sex ratio: no statistically significant differences between treated and  control
- Grossly visible abnormalities: no statistically significant differences  between treated and control
- External abnormalities: no statistically significant differences  between treated and control
- Soft tissue abnormalities: no statistically significant differences  between treated and control
- Skeletal abnormalities (control, 144, 289, 664 mg/m3): 24/106, 26/120,  31/111, 36/110   
no statistically significant differences between treated and control. 
- Other examinations: Three instances of exencephaly were noted in mouse  fetuses (no data on doses). These effects are regarded as not to be  
compound-related by the authors.
Dose descriptor:
NOAEL
Effect level:
>= 664 mg/m³ air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

NOAEL (maternal): based on reduced body weight




Conclusions:
In this inhalation teratogenicity study with mice, isophorone elicited a clinical effect in the pregnant dams in the form of lower body weights (664 mg/m3, day 18 of gestation). During the conduct of the probe study there were three instances of exencephaly noted in mouse fetuses. Based on the
observations made in this study the authors do not believe that these anomalies were related to the test material.
Within the framework of the dose levels and test methods used, it is concluded, that isophorne was not teratogenic or fetotoxic in mice.
Executive summary:

Pregnant CD-1 mice were treated daily for 6 hours by inhalation (whole body) with isophorone from day 6 to day 15 of gestation to examine potential developmental toxicity effects of the test substance. Dosages used were 0 (conditioned air control), 144, 289 and 664 mg/m3, respectively (22 animals per dose level).

Body weight was reduced in mice of the highest dose group (gestation day 18, corrected for uterine weight: -5.6 %). Additionally, in one animal a dose related increase in alopecia was observed, as well as a discoloration of the cervical and aogenital region. Adverse effects on the fetuses were not observed.

Therefore, under the conditions of this inhalation teratogenicity study with mice, the NOAEL for maternal toxicity was determined to be 289 mg/m3 (based on < 6 % reduction in body weight gain). Isophorone was neither embryotoxic nor teratogenic up to the highest test concentration of 664 mg/m3 isophorone.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015-10-22 to end of December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study without restriction
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: EPA OPPTS870.6300
GLP compliance:
no
Remarks:
All techniques and procedures were performed according to the standard operating procedures.Instruments and facilities are routinely supervised and validated according to GLP guidelines.
Limit test:
no
Species:
rat
Strain:
other: Fischer 344 (F344/HanZtn Rj) and Wistar (Crl:Wi(Han))
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Sex: Female
- Strain: Fischer 344 (F344/HanZtn Rj) and Wistar (Crl:Wi(Han))
- Source Fischer 344: Janvier, Route des ChénesSecs, Le Genest Saint-Isle, F-53940 France
- Source Wister: Charles River C/Argenters 7, Local AB 08290 Cerdanyola del Vallés Barcelona – Spain
- Age: 10 - 11 weeks on arrival
- Body weight on day 1 of gestation: Fischer 344: 165.0 - 196.4 g, Wistar: 225.7 - 285.1 g
- Animals per cage (before / after distribution): 5 (1 after distribution and before mating)
- Bedding material: Souralit C16 / 11 (irradiated), Souralit, Spain
- Diet: ad libitum, Global diet, Harlan Teklad Station Road Blackthorn, Bicester Oxon, OX25 1TP United Kingdom
- Water: ad libitum, Tap water
- Acclimatisation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 30 - 70 %
- Light cycle: 12:12 – 07.00 to 19.00 CET
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Remarks:
Sham filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past, nose-only exposure system. Exposure chambers type EC-FPC-232
(anodized aluminium, volume inside compartment: approximately 3 L, equipped with glass exposure tubes were used.
- Method of holding animals in test chamber: The rats were individually exposed in glass tubes matching their size.
- Source and rate of air: Compressed air.
The flow of air at each tube was between 0.5 to 1 L/min, which was sufficient to minimize re-breathing of the test item vapour as it is more than twice the respiratory minute volume of rats.
- Method of conditioning air:
- System of generating particulates/aerosols: Vapors of test item were generated using a VOC (Volatile organic compound) vapor generator
(TSE-systems).
- Temperature, humidity, pressure in air chamber:
Mean chamber temperature was maintained within the recommended range of 22±3°C over the entire exposure period.
Mean chamber relative humidity was maintained within the a range of 14-20% over the entire exposure period, excluding chamber corresponding
to sham air exposed groups (A and D) on day 1 of exposure which showed a relative humidity of 9%.
Throughout the exposure period the oxygen concentration was maintained above 19% (values ranged between 20.8-20.9%) while the CO2
concentration remained below 1% (0.04-0.09%).
- Air flow rate: The airflow rate was maintained at 25 L/min for sham air and low dose exposure chambers over the 18 days of exposure. The
airflow rate for high dose exposure chambers (days 1 and 2 of exposure) was maintained at 50 L/min.
- No diet or drinking water was available during exposure.


TEST ATMOSPHERE
- Brief description of analytical method used:
Vapour samples were captured in a solvent trap containing a defined volume of methanol (cooled with ice) positioned in an empty port of the
inhalation chamber. Based on the results from the technical trials, an additional second solvent trap might be placed immediately after the first
solvent trap to ensure that all test item was captured during sampling. The sampling flow rate matched the air flow rate per port. The defined volume of methanol in the solvent trap was refilled to the start volume after sampling completion. Aliquots of samples of the resulting solution of test item
in methanol were appropriately labelled and stored at 2° - 8°C until analysis or shipment. Test item concentrations were analyzed using an analytical method validated in compliance to GLP at Swiss BioQuant AG

VEHICLE (if applicable)
- Concentration of test material in vehicle: Technical trials were performed without animals and conducted before the animal phase of the study to
establish the conditions for vapour generation and sampling, which included:
- Determination of the target concentration and/or technical limit. Several tests were performed to establish the highest stable vapour
concentration achievable that could be maintained at least for 6 hours. Vapor concentration was aimed to 2.0 mg/L in order to compare the results with a previous non-GLP reproduction toxicity study. A stable respirable vapour at a maximum concentration of 1.25 mg/L could be achieved.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical determination of the vapour concentration was performed thrice during each day of exposure for each test item group. Additional samples
were analysed if considered necessary.
The quantification of test item was performed by column separation with reversed-phase chromatography followed by detection with UV.


Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) to initiate the nightly mating period, until evidence of copulation was
observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The
day of mating was designated day 0 post coitum (GD0).
Male rats of the same source and strain were used only for mating. These male rats are in the possession of Vivotecnia and are not considered part of the test system.
Duration of treatment / exposure:
- Duration of dosing: From day 6 post coitum (implantation) to day 20 post coitum
- Dosing regimen: 6h/day, Groups B and E were exposed together on the same exposure chamber. The same applies to Groups C and F.
Frequency of treatment:
6h/day
Duration of test:
Caesarean section on day 21
No. of animals per sex per dose:
6 females per group, 18 pregnant females per strain were distributed in 3 groups
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for target concentration selection:
The target concentrations were selected based on toxicity information available and the technical limit for the maximal vapour concentration.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
The animals were examined twice daily for mortality and morbidity.
Any clinical signs, discomfort and mortality were recorded in accordance with the humane endpoints guidance document of the OECD.
Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.
Where the condition of an animal required premature sacrifice, it was euthanized by CO2 asphyxiation..

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations in response to treatment were performed on all animals once daily, approximately 1h after exposure.
Any visible clinical signs, discomfort and mortality were recorded.
Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.

BODY WEIGHT: Yes
Body weight was recorded daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
Food was weighed on days 0, 3, 6, 9, 12, 15, 18 and 21 post coitum.
Data were presented in terms of consumption per animal per day for the periods (days 0 – 3), (3 – 6), (6 – 9), (9 – 12), (12 – 15), (15 – 18) and
(18 – 21).

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
Gross necropsy:
At the scheduled necropsy (day 21 post coitum) females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section.
All females were examined, either at the scheduled necropsy or during the study if death occurs.
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora
lutea count and position of fetuses in the uterus, was performed and the data recorded.
The uteri (and contents) of all females with live fetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites

Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight taken.
Any organ with gross lesions was collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if
considered relevant.
Animals dying during the study or sacrificed for animal welfare reasons were also subjected to a gross necropsy. Similarly, nasal cavity, trachea,
larynx and lungs from animals showing relevant signs of irritation during exposure (i.e nasal bleeding) were collected and preserved in fixation
medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant.
Additionally, lungs from all dams were instilled via trachea with formalin at approximately 30 cm H2O pressure collected and fixed in neutral
phosphate buffered 4% formaldehyde solution for possible further examinations.


Ovaries and uterine content:
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents,
corpora lutea count and position of fetuses in the uterus, was performed and the data recorded.
The uteri (and contents) of all females with live fetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites

Fetal examinations:
Fetal Pathology
Fetuses were removed from the uterus by Caesarean section (C-section), sexed, weighed individually, and examined for gross external abnormalities.External examination of foetuses was performed at the test facility. After external examination, foetuses were assigned with a number and sacrificed
by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique). Approximately one half of the foetuses (foetuses with odd numbers) from each litter were fixed in Bouin’s fixative. Then foetuses were sent in the same fixative medium to the test site AnaPath GmbH within 3 weeks. At the test site the fetuses were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed
examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of
glycerin/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol
(ca.94%), foetuses were sent in the same fixative medium to the test site AnaPath GmbH for further skeletal evaluation. Carcasses were processed
through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining
ossified bone) and aqueous glycerin for preservation and storage [see literature (2)]. The skeletons were examined and all abnormal findings and
variations were recorded. The assessment included, but was not limited to all principal skeletal structures including cranium, vertebral column, rib
cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.
Peer review of at least 10% of the fetal examinations was performed. Fetuses with abnormalities were photographed, when considered appropriate.
The foetal pathology results were provided to the study director by e-mail and a pathology phase report was sent to the study director upon
completion of the study for inclusion in the final report
Statistics:
Provided that the sample size was considered adequate, the following parameters were subject to statistical analysis:
- Body weight
- Food consumption
Analysis of data from two groups:
Test of normal distribution Yes Un-paired t-test
No Mann-Whitney test
Test of equal variance No Un-paired t-test with Welch’s correction

Analysis of data from more than two groups
Test of normal distribution Yes One-way ANOVA Dunnett’s post test (comparison versus control group)
Tukey post test (comparison of all groups)
No Dunn’s post test
Test of equal variance No Dunn’s post test
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Over the first two days of exposure period (corresponding to gestation days 6 and 7), results obtained were indicative of excessive toxicity in animals exposed to the high concentration (1.25 mg/L) of test item (Group C, F344 strain and Group F, Wistar strain). As a result, 4 females from group C in addition to 4 females from group F were found dead or sacrificed for humane reasons over this period.
On the basis of these results, it was decided for animal welfare reasons to decrease the target concentration of both experimental groups (Groups C
and F). Thereby, from GD 8 to GD20 the 2 surviving females from each group (Group C, animals ID 17 and 18 and Group F, animals ID 35 and 36) were exposed to the low concentration (0.85 mg/L) being administered as well to groups B (Strain F344) and E (Strain Wistar).
Dose descriptor:
NOAEL
Basis for effect level:
other: maternal toxicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Basis for effect level:
other: developmental toxicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Effect level:
150 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
150 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
see below
Abnormalities:
not specified
Developmental effects observed:
not specified

Maternal data

Maternal mortality

 

Strain

Group

Treatment

Target concentration (mg/L)

Animal ID

Exposure day 1

Exposure day 2

GD8

GD6

GD7

F344

C

Isophorone

1.25

13

FD 1h post dose

 

 

14

 

S pre-dose

 

15

S 5 hs exposure

 

 

16

FD 6 hs exposure

 

 

Wistar

F

Isophorone

1.25

31

 

 

S pre-dose

32

 

 

S pre-dose

33

S 1h post dose

 

 

34

 

 

S pre-dose

FD: Found dead. S: Humane sacrifice

 

Pregnancy rates

F344 Strain: The pregnancy rate for control animals (Group A) was 100%. In the case of the group B (0.85 mg/L), 1/6 animals showed no implantation sites upon c-section, being pregnancy rate 83.3%. Finally, pregnancy rate achieved by the 2 surviving animals of group C (1.25/0.85 mg/L) was 100%.

Wistar Strain: Pregnancy rate achieved in the three experimental groups was 100%.

Clinical signs and observations

F344 Strain: The main and more frequent clinical signs present in animals belonging to all experimental groups were the presence of chromodacryorrhea (eye red secretion, or Harderian gland secretion) piloerection and wet fur immediately after the end of the daily exposure period. The incidence of these clinical signs was slightly higher in test item exposed groups (B and C, 0.85 mg/L) when compared to control animals from this strain.

Wistar Strain: The main clinical signs evident in females belonging to group D (Wistar rats, control group) from GD6 to GD13, comprised the presence of piloerection, chromodacryorrhea together with dirty fur. From GD14 onwards of exposure period, the only remarkable observation found was the presence of dirty fur. Regarding females belonging to group E (test item, 0.85 mg/L) and F (test item, 1.25/0.85 mg/L), piloerection, chromodacryorrhea and dirty fur were recorded over the entire exposure period. The incidence of these signs observed in animals from group E was slightly higher than the observed in the sham control group.

Comparison of findings in both strains: When comparing clinical signs observed in sham control animals from both strains, F344 rats seemed to be more sensitive to the stress induced by the experimental procedures, since the incidence of the main clinical signs observed over the exposure period (piloerection, chromodacryorrhea and dirty fur) was slightly higher in F344 strain.

Maternal body weight and body weight-gain

F344 Strain: No statistically significant differences were observed among groups in body weights and body weight gains prior to the start of the exposures (GD 0-6). Thereafter, over the early gestation period (GD6 -12) body weight loss was observed in the two experimental groups exposed to test item (Group B, 0.85 mg/L and C 1.25/0.85 mg/L), without statistically significant differences being observed with respect to control group.

Thereafter from GD9 to GD21 in the case of animals from group B (0.85 mg/L) maternal body weight gain was still significantly reduced when compared to control animals. As a result, mean body weight gain for the overall exposure period (GD6-GD21) was statistically lower in animals from group B (reaching a body weight gain of -10.13%) when compared to control group (which achieved a body weight gain of 23.18%). This effect was considered to be attributable to maternal toxicity, leading to effects on embryonic development.

Regarding animals from group C (1.25/0.85 mg/L), lower body weight gain was noted for the remainder exposure period (GD9-GD21) for the two surviving animals compared to control group. Hence, mean body weight gain for the overall exposure period (GD6-GD21) in animals from group C reached nearby the 66% of the achieved by control animals. Given to the limited group size in group C statistical analysis could not be performed.

Wistar Strain: Maternal body weight and weight changes were equivalent across all groups prior to the start of exposures (GD0-GD6) as well as over the exposure period (GD6-GD21), with no statistically significant differences being found among the experimental groups.These results were in accordance with c-section observations, which revealed a 100% of dams with viable foetuses in control and test item exposed groups G (Group B, 0.85 mg/L and C 1.25/0.85 mg/L).

Comparison of findings in both strains: When comparing body weight changes of control animals (Group A, strain F344 and group D, strain Wistar) a similar trend was observed in both strains over the entire observation period. In particular, control animals from both strains exhibited body weight gain prior to the start of the exposures (GD 0-6), reaching on GD6 a 5% and 12% increase with respect GD0 in F344 and Wistar control rats, respectively.

Initiation of exposure led to a decrease of body weight in both strains of similar magnitude. Thus, from GD6 to GD9 a body weight drop nearby a 4.3% was observed in animals belonging to control group A (strain F344). Similarly, over this period (GD6-GD9) animals from control group D (strain Wistar) exhibited a body weight loss up to a 3%.

Thereafter, from GD9 to GD21 body weight increase was observed in both experimental groups, being slightly higher in Wistar rats in terms of percentage of increase across the exposure period (16% F344 control rats vs 25% Wistar control rats), as well as over the entire gestation period (23.2% F344 control rats vs 38.74% Wistar control rats).

Food consumption

F344 Strain: Over the pre-exposure period (GD0-GD6) food consumption was similar across all the experimental groups. Afterwards, in general terms, food consumption of females exposed to test item

(Group B, 0.85 mg/L and group C, 1.25/0.85 mg/L) was slightly lower than the observed from sham control F344 rats (Group A) over the entire exposure period. This effect was more remarkable from GD6 to GD9, stage at which animals from test item exposed groups exhibited a decline in food intake up to a 29.5% when compared to control group. Thereafter, from GD9 to GD 21, the drop of food consumption in females exposed to test item (Groups B and C) ranged from 10% to 17%. This effect did not reach statistical significance.

Wistar Strain: Over the pre-exposure period (GD0-GD6) food consumption was similar in control (Group D) and test item exposed (Group B, 0.85 mg/L and group C, 1.25/0.85 mg/L) Wistar rats. Thereafter, exposure to test item did not seem to have an impact in food consumption of any experimental group. Only animals from group E (test item, 0.85 mg/L) exhibited a transient and statistically significant decline in food consumption from GD15 to GD18 (12% respect to control group).

Comparison of findings in both strains: According to the results disclosed in this section, animals from F344 strains seemed to be more sensitive to the exposure to test item in terms of food consumption. This lower food consumption values in F344 strain correspond with body weight effects in these groups together with c-section findings.

Female necropsy data and pregnancy outcome

Macroscopic observations No test item related macroscopic observations were recorded in any animal from any experimental group upon macroscopic necropsy performed on GD21.

Uterine implantation data

F344 Strain: Mean number of corpora lutea was similar across groups. Concerning mean implantation sites and pre-implantation loss, similar results were observed in control animals and in the two animals from group C surviving up to GD21. In particular mean number of implantation sites per animal observed in group A were 10.8, while in group C (1.25/0.85 mg/L) mean number of implantation sites per animal was 10.0. These results involved a mean percentage of pre-implantation loss of 4.7% in control animals (Group A) and equal to 30.5% in group C (1.25/0.85 mg/L).

In the case of group B (0.85 mg/L), mean percentage of pre-implantation loss was 42.4%, being remarkably higher than the 4.70% recorded in control animals. It should be noted that one female belonging to this group (ID11) showed a 100% of pre-implantation loss together with the circumstance that within group variability was large. Accordingly, this effect did not achieve statistical significance.

In terms of post-implantation losses, a clear treatment related effect was observed in groups B (0.85 mg/L) and C (1.25/0.85 mg/L). In particular, mean post-implantation losses per animal in group B was 84.4% while in the case of group C this value reached a 63.7%. These values were remarkably higher than the 17.52% of mean post-implantation losses per animal found in the control group, with no statistically significant differences being observed.

It must be pointed out that, while in control group the major part of the post-implantation losses were classified as early or embryonic resorptions (11/12 losses) in the case of test item exposed animals, most of these losses took place at an earlier stage. In particular, in group B (0.85 mg/L) 24 of the 27 post-implantation losses recorded were defined as empty implantation site, meaning a very early resorption or aborted implantation. Concerning group C, animal ID 17 showed a 100% of post-implantation losses, all of them considered empty implantation sites, whereas animal ID 18 showed 3 post-implantation losses (from a total of 11 implantation sites), being the 3 described as embryonic resorptions.

All together, these circumstances led to a statistically significant decrease in the number of viable foetuses recorded in animals from group B (0.85 mg/L) when compared to control group. In particular, a total of 56 live foetuses were observed upon c-section in animals from group A, while in the case of group B only 1 female presented viable fetuses (a total of 5).

Wistar Strain: Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation losses were similar across all groups. Moreover, post-implantation losses and litter sizes were similar when comparing test item exposed and control animals.

Comparison of findings in both strains: In general terms, comparison of data from control groups showed a similar reproductive performance for both strains concerning to mean pre-implantation losses per animal. Thereafter, F344 females showed a worse reproductive performance compared to Wistar rats, when considering the mean percentage of post-implantation losses per animal (17.52% in F344 strain versus 6.56% in Wistar strain). On the other hand, litter size was higher in Wistar rats (12.8 fetus per litter) than in F344 strain (8.8 fetus per litter).

These results are in agreement with the published literature, reporting relatively poor reproductive performance of the F344 strain (inbred strain) when compared to outbred strains such as Wistar rats, known for their better reproductive capabilities. Therefore, F344 strain would not be the appropriate model of choice for prenatal developmental toxicity studies.

Gravid uterine weight data

F344 Strain: Upon c-section the 6 females belonging to control group (Group A) were determined to be gravid. By contrast, in the case of group B (Test item, 0.85 mg/L) from the 6 animals surviving until GD21 only 1 (ID7) was found to be gravid on GD21 and showed viable fetuses. Finally, regarding from the two females from group C (Test item 1.25/0.85 mg/L) surviving the entire observation period, only one of them (ID18) was determined to be gravid and showed viable festuses.

Therefore, on the basis of the limited group size available, no remarkable differences were found in gravid uterine weight between control and test item exposed groups or neat terminal body weight. A decline in neat body weight gain from GD6 to GD21, was found in all experimental groups, ranging between -7.7% to -9.5%.

Wistar Strain: Upon c-section conducted on GD21, the 6 females from groups D (Control group) and group E (Test item, 0.85 mg/L) were determined to be gravid. In addition, the two surviving females from group F (Test item, 1.25/0.85 mg/L) well found as well to be gravid.

Gravid uterine weight, neat terminal body weight together with neat body weight gain was very similar among the three experimental groups, with no statistically significant differences being observed. A decline in neat body weight gain from GD6 to GD21, was found in all experimental groups, ranging between - 4.2 % to - 4.9%.

Comparison of findings in both strains: Control animals from both strains showed a decrease in neat body weight gain from GD6 to GD21, being this effect more noticeable in F344 rats. In particular, over the exposure period F344 control females exhibited a neat body weight gain up to a -9.4%, while in Wistar rats neat body weight gain was - 4.2%.

Conclusions:
Fischer 344 strain:
Based on the results of this study, a no observed-adverse-effect level (NOAEL) for maternal effects and embryo-fetal development could not be established for this strain.
Wistar strain:
On the basis of these results, it was considered that a no observed-adverse-effect level (NOAEL) for maternal effects and embryo-fetal development was evident at 0.85 mg/L (150 ppm).
Strain comparison:
- The F344 rat is an inbred strain which was used for years as a suitable animal model for reproduction and development
toxicity studies. Nevertheless, over time these animals developed several health issues inherent to the strain, involving among
others decreased fecundity (King-Herbert and Thayer 2006), reasons why their use within this type of studies was
discouraged. By contrast, outbred strains such as Wistar rat are known for their hybrid vigor, large litter size, long
lifespan, resistance to disease, and low neonatal mortality (Hedrich 2006).
- In the present study, F344 rats exposed to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from
gestation day 6 to gestation day 20 showed an exposure related effect on pregnancy outcome, in terms of pre and
post-implantation loss. By contrast, such effects were not observed in Wistar rats following the same exposure regimen.
Thereby, inherent strain reproductive performance characteristics may have enhanced the test item effect on
embryo-fetal development observed in F344 rats.
- Results of the present study together with the information existing in the published literature, provide clear evidence that F344
rats are not a suitable strain for prenatal developmental toxicity studies.
Executive summary:

Objective

In a first instance this study was designed to assess the potential effects of the test item on pregnancy and embryo-fetal development in F344 and Wistar rats, following daily inhalation exposure (nose-only) at concentrations of 0.85 (150) and 1.25 mg/L (220 ppm), during 6 hours per day, from implantation to one day prior to scheduled caesarean section (day 6 to day 20 post-coitum, inclusive). Therefore this study aimed to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

Furthermore, these results should serve as the basis for the selection of the exposure concentrations to be used in a further regulatory prenatal and developmental toxicity study to be performed according to guideline OECD 414.

Study Design

For these purposes, 18 pregnant females per strain were distributed in 3 groups (6 animals per group). F344 rats were allocated into groups A-C. Animals from group A were exposed to filtered air (Group A, sham controls), whereas animals allocated in group B were exposed to the low concentration of the test item (0.85 mg/L) and animals from group C were exposed to the high concentration

(1.25 mg/L).

Similarly, Wistar rats were allocated into groups D-F. Group D served as sham control, while animals from groups D

and E were exposed to the low (0.85 mg/L) and high (1.25 mg/L) doses of test item, respectively.

Over the first two days of exposure (Gestation day 6-7), the high concentration of the test item was reduced from 1.25 mg/L to 0.85 mg/L (low concentration) due to unscheduled mortality and adverse clinical signs observed at this exposure concentration in both strains.

All animals were observed twice daily for mortality and clinical observations. Body weights, and food consumption were recorded at appropriate intervals. On GD 21, a cesarean section was performed on each female and selected organs were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Study Results

F344 strain

Over the first two days of exposure (GD6-GD7), 4 females from the high concentration group (Group C, 1.25 mg/L) were found dead (2/4) or sacrificed for humane reasons (2/4). Thus, only 2/6 animals from this group survived until the end of the exposure period, involving exposure to a concentration of 1.25 mg/L from gestation day (GD) 6 to GD 7 and to a concentration of 0.85 mg/L thereafter, from GD8 to GD20.

Exposure related clinical signs (such as piloerection and wet fur) were found in all experimental groups, which a slight higher incidence in test item treated animals. Over the early pregnancy (GD6-GD12) no statistically significant differences were observed in body weight or body weight gain between sham control and test item exposed animals. Thereafter, from GD9 to GD121 a statistically significant body weight reduction was observed in females from group B (0.85 mg/L). This effect was considered to be attributable to maternal toxicity, leading to effects on embryonic development.

In general terms, food consumption of test item exposed females tended to be slightly lower of the observed for sham control animals over the entire observation period. Nevertheless, these differences did not achieve statistical significance.

No test item related macroscopic findings were noted in animals from group B and C. A test item related effect was observed regarding pre and post-implantation losses in animals from both experimental groups (0.85 and 1.25 / 0.85 mg/L), which showed remarkably higher percentages of losses with respect to control group.

Upon c-section (GD21) 5/6 females from group B and 2/2 females from group C showed implantation sites and/or viable fetuses. On the basis of the limited group size available, no remarkable differences were found in gravid uterine weight or in neat body weight gain (GD6-GD1) when comparing test item treated animals with sham control groups.

Finally, there was no effect of exposures in fetal weight at GD21 and no external, visceral or skeletal abnormalities or variations were noted.

Wistar strain

Over the first two days of exposure (GD6-GD7), 4 females from the high concentration group (Group F, 1.25 mg/L) were sacrificed for humane reasons. Thus, only 2/6 animals from this group survived until the end of the exposure period, involving exposure to a concentration of 1.25 mg/L from gestation day (GD) 6 to GD 7 and to a concentration of 0.85 mg/L thereafter, from GD8 to GD20.

No test item exposure related effects were observed on maternal observations, gestational parameters or fetal evaluations.

Conclusion

Taken together the results disclosed in the present report, it can be concluded that under these experimental conditions:

F344 strain

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 1.25 mg/L (220 ppm), 6 hours/day

from gestation day 6 to gestation day 7, resulted in maternal toxicity, which involved clinical signs of toxicity and mortality.

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L (150 ppm), 6 hours/day

from gestation day 6 to gestation day 20, did induce maternal toxicity (statistically significant body weight loss).

- Accordingly, a no observed-adverse-effect level (NOAEL) for maternal effects could not be established in this study.

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from gestation day 6

to gestation day 20 led to an exposure related effect on pregnancy outcome, in terms of pre and post-implantation loss.

These effects are considered to be due to overt maternal toxic effects. On the basis of the very limited analysis performed under

the conditions of this study, due to the low number of foetuses available in females exposed to test item vapour by inhalation

at 0.85 mg/L, exposure was not associated with any effect on fetal weight or the incidence of fetal abnormalities and variants.

- On the basis of these results, a NOAEL for embryo-fetal effects could not be established for this strain.

Wistar strain

- Exposure of Wistar pregnant rats to test item vapour by inhalation (nose-only) at 1.25 mg/L (220 ppm), 6 hours/day

from gestation day 6 to gestation day 7, resulted in maternal toxicity, which involved clinical signs of toxicity and mortality.

- Exposure of Wistar pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L (150 ppm), 6 hours/day

from gestation day 6 to gestation day 20, did not induce maternal toxicity and did not have any effect in pregnancy outcome,

in terms of pre- or post-implantation loss, fetal weight or the incidence of fetal abnormalities and variants.

- On the basis of these results, it was considered that a no observed-adverse-effect level (NOAEL) for maternal effects

and embryo-fetal development was evident at 0.85 mg/L (150 ppm).

Strain comparison

- The F344 rat is an inbred strain which was used for years as a suitable animal model for reproduction and development

toxicity studies. Nevertheless, over time these animals developed several health issues inherent to the strain, involving among

others decreased fecundity (King-Herbert and Thayer 2006), reasons why their use within this type of studies was

discouraged. By contrast, outbred strains such as Wistar rat are known for their hybrid vigor, large litter size, long

lifespan, resistance to disease, and low neonatal mortality (Hedrich 2006).

- In the present study, F344 rats exposed to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from

gestation day 6 to gestation day 20 showed an exposure related effect on pregnancy outcome, in terms of pre and

post-implantation loss. By contrast, such effects were not observed in Wistar rats following the same exposure regimen.

Thereby, inherent strain reproductive performance characteristics may have enhanced the test item effect on

embryo-fetal development observed in F344 rats.

- Results of the present study together with the information existing in the published literature, provide clear evidence that F344

rats are not a suitable strain for prenatal developmental toxicity studies.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Species:
rat
Quality of whole database:
guideline comparable inhalation teratogenicity test; Test procedure in accordance with generally accepted scientific standards and described in sufficient detail; Klimisch 1 (reliable without restriction)
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the first inhalation teratogenicity study with rats, isophorone elicited a clinical effect in the pregnant dams in the form of decreased food consumption (111 ppm (640 mg/m3), days 6-20 and 0-20), lower body weights (111 ppm 640 mg/m3, days 12 and 15 of gestation), and dose related increases in alopecia and staining of the cervical and anogenital areas. During the conduct of the DRF study (Exxon, 1983) there was one instance of exencephaly noted in a rat fetus at 144 ppm. Based on the observations made in this study the authors do not believe that this anomaly was related to the test material. Within the framework of the dose levels and test methods used, it is concluded, that isophorone was not teratogenic or fetotoxic in rats (Exxon, 1984).

In the second inhalation teratogenicity study with mice, isophorone elicited a clinical effect in the pregnant dams in the form of lower body weights (111 ppm (640 mg/m3), day 18 of gestation). During the conduct of the DRF study (Exxon, 1983) there were three instances of exencephaly noted in mouse fetuses at 144 ppm. Based on the observations made in this study the authors do not believe that these anomalies were related to the test material. Within the framework of the dose levels and test methods used, it is concluded, that isophorone was not teratogenic or fetotoxic in mice (Exxon, 1984).

DRF for OECD 414 (inhalation), F344 and Wistar rats (Vivotecnia, 2016)

Test strategy

- Choice of the test (rat) strain:

ECHA specially asked for the use of F344 rats in the OECD 414 study in order to compare the results with the previous inhalation teratogenicity study (Exxon, 1984), where this strain was used (ECHA Decision, 2015).

The Registrants decided to perform the prenatal developmental toxicity study (inhalation, rat) (OECD 414) as a limit test as requested by ECHA, but to use two difference strains of rats (requested F344 rat and additionally Wistar rat) due to the suitability of the Fischer rat strain. Due to the fact that no historical controls are available F344 rats and the differences in timing of developmental events compared to strains like Wistar which are commonly tested in developmental/reproductive toxicity studies, the assessment of results is hardly possible. These two points do not allow to differentiate between strain specific and/or incidental findings and substance related effects. For this reason EPA guideline explicitly points out that Fischer 344 strain should not be used for developmental studies (see EPA Health effects Test Guidelines OPPTS 870.6300, August 1998). Therefore, in this study Wistar rats were used for quality control and comparison of findings in both strains and with available historical control data from Wistar rats.

- Choice of concentration:

The Registrants decided to perform a dose-range finding study in advance to estimate the maximum tolarable and attainable concentration as requested in the ECHA decison.

In the decision ECHA pointed out: " The choice of the concentrations tested in the main studies in rats and mice (Exxon, 1984) is questionable since no major toxicity was noted in dams at 144 ppm in the range-findng study (Exxon, 1983) and this does not justifiy lowering the concentrations." Therefore, the Registrants decided to conduct the DRF study with target concentrations of 150 and 220 ppm. The estimation for the technical limit for the maximal vapour concentration is based on the physico-chemical properties of the test material. The actual technical limit was conducted to be at 220 ppm during technical trails and was used at high dose.

In the new DRF study (Vivotecnia, 2016) a high and promptly mortility was observed in F344 rats at the high dose (220 ppm) as well as a statistically significant lower body weight and body weight gain was observed at low dose (150 ppm) when compared to air-treated F344 rats (control group). Both findings are clear maternal effects.

In the decision ECHA pointed out: "However, it cannot be concluded that the effect is a chance event because no concentration above 144 ppm was tested. This concentration was not associated with overt maternal toxicity and therefore an additional assay using the maximum tolerated and attainable concentration is needed." In the new DRF study (Vivotecnia 2016) isophorone vapour with approx. 150 ppm and 220 ppm were generated: Both concentration was associated with overt maternal toxicity in F344 rats but no major toxicity was observed in Wistar rats. For animal welfare reason the high dose exposure (220 ppm) was stopped for both strains. However, this means that 150 ppm is the maximum tolerated concentration while 220 ppm is the maximum attainable concentration for F344 rats. No test item-related abnormalities were noted during the visceral or skeletal examinations of fetuses of both treated strains (F344 and Wistar rats). 

The legal basis for this test strategy is the ECHA Factsheet "Follow up to dossier evaluation decisions p.2; ECHA-13 -FS-05"

Results of the study:

F344 strain

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 1.25 mg/L (220 ppm), 6 hours/day

from gestation day 6 to gestation day 7, resulted in maternal toxicity, which involved clinical signs of toxicity and mortality.

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L (150 ppm), 6 hours/day

from gestation day 6 to gestation day 20, did induce maternal toxicity (statistically significant body weight loss).

- Accordingly, a no observed-adverse-effect level (NOAEL) for maternal effects could not be established in this study.

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from gestation day 6

to gestation day 20 led to an exposure related effect on pregnancy outcome, in terms of pre and post-implantation loss.

These effects are considered to be due to overt maternal toxic effects. On the basis of the very limited analysis performed under

the conditions of this study, due to the low number of foetuses available in females exposed to test item vapour by inhalation

at 0.85 mg/L, exposure was not associated with any effect on fetal weight or the incidence of fetal abnormalities and variants.

- On the basis of these results, a NOAEL for embryo-fetal effects could not be established for this strain.

Wistar strain

- Exposure of Wistar pregnant rats to test item vapour by inhalation (nose-only) at 1.25 mg/L (220 ppm), 6 hours/day

from gestation day 6 to gestation day 7, resulted in maternal toxicity, which involved clinical signs of toxicity and mortality.

- Exposure of Wistar pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L (150 ppm), 6 hours/day

from gestation day 6 to gestation day 20, did not induce maternal toxicity and did not have any effect in pregnancy outcome,

in terms of pre- or post-implantation loss, fetal weight or the incidence of fetal abnormalities and variants.

- On the basis of these results, it was considered that a no observed-adverse-effect level (NOAEL) for maternal effects

and embryo-fetal development was evident at 0.85 mg/L (150 ppm).

Strain comparison

- The F344 rat is an inbred strain which was used for years as a suitable animal model for reproduction and development

toxicity studies. Nevertheless, over time these animals developed several health issues inherent to the strain, involving among

others decreased fecundity (King-Herbert and Thayer 2006), reasons why their use within this type of studies was

discouraged. By contrast, outbred strains such as Wistar rat are known for their hybrid vigor, large litter size, long

lifespan, resistance to disease, and low neonatal mortality (Hedrich 2006).

- In the present study, F344 rats exposed to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from

gestation day 6 to gestation day 20 showed an exposure related effect on pregnancy outcome, in terms of pre and

post-implantation loss. By contrast, such effects were not observed in Wistar rats following the same exposure regimen.

Thereby, inherent strain reproductive performance characteristics may have enhanced the test item effect on

embryo-fetal development observed in F344 rats.

- Results of the present study together with the information existing in the published literature, provide clear evidence that F344

rats are not a suitable strain for prenatal developmental toxicity studies.

Discussion and conclusions:

This DRF study was performed in F344 rats using a maximum tolerable concentration of 150 ppm as well as a maximum attainable concentration of 220 ppm. Due to the difficulties with F344 rats (see above) Wistar rats were tested also in the DRF study in order to be able to generate scientifically established results. Due to the overt maternal toxicity reproductive effects (post implantation loss) were observed in F344 rats but not in Wistar rats. No test-item related abnormalities were noted during the visceral or skeletal examinations of foetuses of both strains. In so far the results of the Exxon studies (Exxon, 1984) were not reproducible. In particular against the background of all problems and difficulties with Fisher rats and due to animal welfare reasons it make no sense to perfom a main OECD 414 study with this strain because no scientifically based results can be generated. Additionally, no developmental effects were reported at lower concentration (111 ppm) in the Exxon study (Exxon, 1984) and therefore, it makes no sense to perform a main study with F344 rats with significantly lower concentrations.

On the basis of the results of the DRF study and the considerations made it is concluded that the registrants have fulfiled ECHA's requirements fully and they have assured that the results by comparing with the Wistar strain allow a scientifically sound interpretation of the results. The legal basis for the performed test strategy is the ECHA Factsheet "Follow up to dossier evaluation decisions

p.2; ECHA-13 -FS-05".


Justification for classification or non-classification

Regarding toxicity to reproduction (embryotoxicity and fertility) the substance isophorone is not classified according to the criteria of CLP Regulation 1272/2008.