1-[2-(allyloxy)ethyl-2-(2,4-dichlorophenyl)-1H-imidazolium hydrogen sulphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reliable, key reproductive toxicity study is available for imazalil sulphate. Therefore, reliable data from the supporting substance imazalil is used to cover this endpoint.

Reproductive toxicity of imazalil was evaluated in a full two-generation study in rats at nominal doses of 0, 5, 20 and 80 mg/kg bw/d (Dirkx et al., 1992b, K2). The highest dose caused parental toxicity as indicated by reduced body weight and body weight gain, increased incidence of pilo-erection, as well as vacuolisation of hepatocytes in P1 males. At this dose, females also showed a reduced gestation rate and an increased duration of gestation. The later was considered responsible for an increased rate of dystocia. Reproductive toxicity manifested at 80 mg/kg bw/d as a slightly reduced number of implantations, a reduced number of live pups and an increased number of stillborn pups. Reduced offspring survival was considered adverse only for the high dose group. Hence, the NOAEL for parental and reproductive toxicity was identical with 20 mg/kg bw/d (nominal).

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Data from the related substance imazalil base is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

parental toxicity

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

80 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

>= 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

80 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F1

Effect level:

>= 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no teratogenic effects reported

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F2

Effect level:

>= 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no teratogenic effects reported

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

No reliable, key reproductive toxicity study is available for imazalil sulphate. Therefore, reliable data from the supporting substance imazalil is used to cover this endpoint.
In the two-generation study (24 M, 24 F Wistar rats) with nominal dietary doses of 0, 5, 20 and 80 mg/kg bw/d Imazalil, parental toxicity was seen at the highest dose (reduced bw and bw gain, increased incidence of pilo-erection and, in P1 males, vacuolisation of hepatocytes). At this dose, a reduced gestation rate and increased duration of gestation were also seen in females, the latter considered responsible for the concurrent increased rate of dystocia. At the highest dose, reproductive toxicity was seen as a slightly reduced number of implantations, reduced number of live pups and offspring survival and increased number of stillborn pups. No teratogenic effects were reported.
The same is assumed to be correct for the target substance.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-01-31 to 1991-09-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: Toxicological principles for the Safety Assessment of Direct Food Additives and Color Additives used in Food - US FDA -Washington DC

Version / remarks:

1982

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: The rules governing medicinal products in the European Community. Volume III: Guidelines on the Quality, Safety and Efficacy of medicinal products for human use.

Version / remarks:

Revised and completed edition, January 1989

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Pesticide assessment guidelines - Subdivision F - Hazard evaluation - Human and domestic animals

Version / remarks:

Revised edition. November 1984

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Guidelines for toxicity of drugs manual - Pharmaceutical Affairs Bureau, Ministry of Health and Welfare - Japan

Version / remarks:

1990

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch No ZE023979G3A 231
- Expiration date of the lot/batch: No data
- Purity test date: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature in closed containers as delivered under the responsibility of a board certified pharmacist
- Solubility and stability of the test substance in the solvent/vehicle: The concentration and stability of the test article in the food were controlled after preparation. The concentration of the test article in the preparation was guaranteed for the period during which the preparation was used. Therefore, it is concluded that the concentration and stability of the test article in the food mixtures complied with the concept of the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: see section diet preparation

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat was selected as the rodent species following review of available data on toxicity and pharmacology and also because of the possibility to compare the hereby recorded data with the historical control data of other similar studies conducted with the same test system in the same testing facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: test facility's permanent non-inbred laboratory colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males: 60 days; Females: 2-3 months; (F1;F2) from birth
- Weight at study initiation: (P) Males: 290.8-290.9 g; Females: 182.2-188.3 g; (F1) Males/Females: 6.6 to 6.9 g; (F2) Males/Females: 6.6 to 6.7 g.
- Fasting period before study: no data
- Housing: air conditioned rooms and placed in wire-mesh cages. Cohabitation ws allowed for maximum 3 weeks in wire-mesh cages between one male and one female, equivalently dosed in the first generation, whereas in the 2nd generation, cohabitation was done between 1 male and 3 females. As soon as sperm was observed, the female was housed individually until the end of the pregnancy
- Diet (e.g. ad libitum): ad libitum ; fresh tap-water in drinking bottles
- Water (e.g. ad libitum): ad libitum, Huybrechts rodent food
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): 18 / day
- Photoperiod (hrs dark / hrs light): no data
IN-LIFE DATES: no data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): prepared freshly
- Mixing appropriate amounts with (Type of food): powder rodent food and vitamin premix
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: 1M/1F for generation 1; 1M/3F for generations 2 and 3
- Length of cohabitation: max 3 weeks
- Proof of pregnancy: The occurence of copulation was established by daily vaginal inspection for sperm. Sperm in the vaginal smear in the morning, arbitrarily dated insemination back to midnight. Thus the day sperm was found was considered to be day 1 of gestation
- Unsuccessful pairing replacement of first male by another male with proven fertility: no data
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): individually until the end of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and stability of the test article in the food were controlled after preparation. The concentration of the test article in the preparation was guaranteed for the period during which the preparation was used. Therefore, it was concluded that the concentration and tability of the test article in the food mixtures complied with the concept of this study.

Duration of treatment / exposure:

Parents:
- males: 60 days prior to and during mating period
- females: 60 days prior to and during mating and during pregnancy and the weaning period
First and second generation: during growth into adulthood, mating, pregnancy until weaning of the second generation

Frequency of treatment:

daily and continuously until weaning of the second generation

Details on study schedule:

- F1 parental animals not mated until 3 months after selected from the F1 litters.
- Age of the pups when selection of parents from F1 generation: no data
- Age at mating of the mated animals in the study: 3 months + 3 weeks max (mating period)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Actual test article intake during the pre-cohabitation period: 4.2 and 5.0 mg/kg bw/day for males and females
Actual test article intake during pregnancy period (first generation): 4.0 mg/kg bw/day
Actual test article intake during pregnancy period (second generation): 4.3 mg/kg bw/day

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Actual test article intake during the pre-cohabitation period: 17.6 and 21.5 mg/kg bw/day for males and females
Actual test article intake during pregnancy period (first generation): 16.3 mg/kg bw/day
Actual test article intake during pregnancy period (second generation): 18.6 mg/kg bw/day

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

Actual test article intake during the pre-cohabitation period: 70.5 and 104.3 mg/kg bw/day for males and females (In females, the higher actual dosage rate in the 80 mg/kg bw/day group does not really reflect the real test article intake, since increased waste of food was evidenced in this group.)
Actual test article intake during pregnancy period (first generation): 86.8 mg/kg bw/day (the exact test article intake is considered to be lower due to waste of food)
Actual test article intake during pregnancy period (second generation): 87.8 mg/kg bw/day (waste of food resulted in an apparently higher actual test article intake).

No. of animals per sex per dose:

24 males and 24 females per group, 4 groups

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based upon previously conducted toxicity studies
• Exp 342: rats were dosed during 3 months at 5, 20 and 80 mg/kg bw/day. No adverse effects were noted at 5 mg/kg bw/day whereas dosing at 20 mg/kg bw resulted in altered blood, serum and urinary parameters. At 80 mg/kg bw/day also histological liver changes were found.
• Exp 480: rats were dosed at the same dose level as above. Dosing at 5 mg/kg bw/day did not result in adverse effects whereas dosing at 20 and 80 mg/kg bw/day dosed during 12 and 24 months were considered as slightly toxic dose levels. Altered blood parameters and histological liver changes were present at 20 mg/kg bw/day, whereas at 80 mg/kg bw/day also altered serum and organ weight variables were noted.
• Exp 581: rats were dosed at 2.5, 10 and 40 mg/kg bw/day during either 6, 18 or 30 months. No adverse effects were found at 2.5 mg/kg bw and 10 mg/kg bw/day. At 40 mg/kg bw/day, adverse effects on body weight, organ weight and histological changes were observed.
• Exp 2003: rats were dosed from day 6 until day 16 of pregnancy at doses of 40, 80 and 120 mg/kg bw/day. At all dose levels decreased food consumption and body weight were noted. At 80 and 120 mg/kg bw also the weight of the pups decreased. An increased resorption rate was noted at 120 mg/kg bw/day only.
• In a peri-and postnatal study (Exp 597), no adverse effects were noted at 5 and 20 mg/kg bw/day. At 80 mg/kg bw/day, food consumption and survival rate were decreased while an increase in dead pups was noted.
• In a previously conducted 3 generation study in rats (Exp 736) doses were 5, 20 and 80 mg/kg bw/day during 3 generations. No adverse effects were noted at all dose levels. This study was judged to be repeated mainly because histopathology was not performed and also because the dosing schedule in parent animals was too short.
In the present study, the dose of 80 mg/kg bw/day was determined as the high doe whereas 5 and 20 mg/kg bw/day were chosen as low and medium dose levels. Animals were dosed at least 60 days prior to mating and histopathology was done.
- Rationale for animal assignment (if not random): no data
- Fasting period before blood sampling for clinical biochemistry: no blood samples taken

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes (e.g. general condition, chromodacryorrhea, circling animal, piloerection, red stained on external nares, haircoat, skin irritation, subcutaneous mass, food waste)
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Determined for males and females during the 60 days of the pre-cohabitation period and for all females on day 1 and 22 of the presumed pregnancy (days after copulation) and during a 3-week lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, determined for males and females during the 60 days of the pre-cohabitation period and for all females on day 1 and 22 of the presumed pregnancy (days after copulation) and during a 3-week lactation period
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

not investigated

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations:
testes, epididymides and accessory male genital sex glands (control and 80 mg/kg bw/day dosed animals).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no data
- The selection procedure was designed so that each female which delivered a litter was represented by at least one male and one female pup. The parentage of each litter was recorded so that brother-sister mating in the 2nd generation was avoided.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number of live/dead pups / female, mean litter size, number of implantations, bw of pups at birth and day 4, body weigth of males/females day 14, bw of males/females day 21, survival rate after 4, 14 and 21 days.

GROSS EXAMINATION OF DEAD PUPS:
no, possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not assessed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not assessed

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, at the end of the mating period.
- Maternal animals: All surviving animals, at the end of lactation.

HISTOPATHOLOGY
The testes, epididymides and accessory male genital sex glands, the kidney and liver of P1 and F1 males and the ovaries, uterus, vagina, the kidney and liver of the P1 and F1 females were trimmed, embedded, sectioned, stained and mounted by a procedure as much standardized as possible. The hematoxylin-eosin staining method was used. The female genital tract organs and the liver and lidneys of all dosage groups were examined, while the male genital tract organs were examined of control and 80 mg/kg bw/day dosed animals.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed (on information on age when sacrified).
- These animals were subjected to a complete gross necropsy.

GROSS NECROPSY
- Gross necropsy was conducted on the following organs: vagina, uterus, ovaries, tested, epididymides, seminal sevicles, prostates, kidneys, liver.

HISTOPATHOLOGY / ORGAN WEIGHTS - no data

Statistics:

The Chi-Square test for pairwise copmarison with control according to Siegel (two-tailed, Yates' correction for continuity, was used as the statistical method for clinical observations, mortality, copulation, fertility and gestation rate, survival rate.
The Mann-Whitney U test for paiwise comparison with control accroding to Siegel (two-tailed, correction for ties) was used as the statistical method for body weight and body weight gain, food consumption, cohabitation-mating interval, duration of gestation, live, dead and resorbed fetuses - mean litter size implantations, anomalies, histopathology.

Reproductive indices:

no data

Offspring viability indices:

no data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During the pre-cohabitation period, the observations were comparable between groups for males. No adverse effects at 5 and 20 mg/kg bw/day.
Increased presence of wate of food during the precohabitation period, pregnancy and lactation period fo the first and second generation at 80 mg/kg bw/day. Increased incidence of dystocia in the first and second generation. In addition, an increased incidence of animals showing pilo-erection (indicative for a decreased physical condition) was present in animals of the first generation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortalities were noted in whatever group in males prior to (60 days) and/or during the mating period (max 3 weeks)
Dosing at 80 mg/kg bw resulted in the following sacrifices:
- female 92 was sacrificed on day 14 of the lactation period. Clinical symptoms were: bad condition, piloerection, hypernea and red vaginal discharge. Autopsy revealed hydrothorax and hydroperitoneum, congestion of the liver and fibroendocartis.
- female 113 was sacrificed on day 1 after parturition with clinical symptomsof bad condition, respiratory dificulties, red stained external nares, chromodacryorrhea and red vaginal discharge. Autopsy revealed catarrhal pneumonia of all lobes and a pale liver.
No test article or dose related mortalities noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pre-cohabitation period:
- No adverse effects at 5 and 20 mg/kg bw/day
- Males: significant decrease weight change was observed at 80 mg/kg bw/day
- Females: similar but less pronounced decrease at 80 mg/kg bw/day

During pregnancy (day 1 to 22):
- No adverse effects at 5 and 20 mg/kg bw/day
- statistically significant decrease in body weight change noted at 80 mg/kg bw.

During the lactation period (3 weeks):
- No adverse effects at 5 and 20 mg/kg bw/day
- significantly decreased weight change noted at 80 mg/kg bw/day throughout the entire lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption:
-during pre-cohabitation period: Comparable in controls and doses animals
-during pregnancy and lactation: comparable between groups in both the first and second generation
Test article intake: the actual intake corresponded well with the proposed dose levels

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No test article related histological changes were noted in the 5 and 20 mg/kg bw/day dosage groups.

Accessory male genital sex glands: comparable findings in the control and the 80 mg/kg bw/day dosed group.
Epididymes: within normal limits
Testes: comparable findings between controls and 80 mg/kg bw/day dosed animals.
Liver: Comparable findings between controls and dosed animals expect for an increased incidence of large vacuoles in the hepatocytes of the 80 mg/kg bw/day in dosed P1 males. This target organ has been observed at the same dose level in previously conducted toxicity studies in rats.

Female genital tract (ovary, uterus and vagina):
In the 80 mg/kg bw/day dosed P1 female rats a decreased number of animals with corpora lutea of lactation was noted. This is a secondary consequence to the increased incidence of stillborn pups and a decrease in survival rate of pups and therefore decreased lactation stimulus at this dose level. The slight increase of metestrus stage as observed in the ovaries and vagina of the 20 mg/kg bw/day dosed P1 females is considered as a coincidental finding. For the uterus all findings were within normal limits.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Pregnancy: No adverse effects at 5 and 20 mg/kg bw/day. A decreased gestation rate is noted at 80 mg/kg in the first generation.
Copulation rate and fertility rates are comparable between groups while a decreased gestation rate is present at 80 mg/kg bw/day.

Duration of gestation:
No adverse effects at 5 and 20 mg/kg bw/day. The duration of gestation is significantly increased at 80 mg/kg bw/day. As a result of this a higher percentage of dystocia was observed in the 80 mg/kg bw/day dosed group.

Cohabitation-mating interval:
Cohabitation-mating intervals remained comparable between groups in both the first and the second generation.

A higher incidence of stillborn pups and associated herewith a lower incidence of live pups was observed at 80 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

parental toxicity

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

80 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortalities were noted in whatever group in males prior to (60 days) and/or during the mating period (max 3 weeks)
Dosing at 80 mg/kg bw resulted in the following deaths and sacrifices:
- female 605 from the control groups died on day 15 of the lactation period after delivering 10 live pups. Autopsy revealed congestion and emphysema of the lungs.
- female 702 of the 80 mg/kg bw/day group died on day 13 of the lactation period, without previous clinical signs. Autopsy revealed endocarditis.

No test article or dose related mortalities noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During pregnancy (day 1 to 22): statistically significant decrease in body weight change noted at 80 mg/kg bw.

During the lactation period (3 weeks): significantly decreased weight change noted at 80 mg/kg bw/day throughout the entire lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

comparable in all groups

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Accessory male genital sex glands: comparable findings in the control and the 80 mg/kg bw/day dosed group.
Epididymes: within normal limits
Testes: comparable findings between controls and 80 mg/kg bw/day dosed animals.
Kidney: In the 20 mg/kg bw/day dosed male rats of the F1 generation, a slight decrease in basophilic tubuli was noted. This finding is considered coincidental.
Liver: Comparable findings between controls and dosed animals

Female genital tract (ovary, uterus and vagina):
Differences in the number of animals with corpora lutea of lactation between control and dosed groups of the female F1-generation were not detected. In all dosed groups of the female F1-generation a decrease in number of corpora lutea generations and a decrease in clear aspect of the interstitial tissue was observed in comparison to the controls. this is due to the individual variation in moment of sleughtering with respect to the weaning of pups. Other statistical differences observed in the ovaries (slight increase of proestrus and of estrus stage) and the uterus (high epithelium and healing of tissue at mesanteric site) of the female F1 generation are occasional findings.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Copulation rate, fertility and gestation rates are comparable between groups.
The duration of gestation is significantly increased at 80 mg/kg bw/day. As a result of this a higher percentage of dystocia was observed in the 80 mg/kg bw/day dosed group.
Cohabitation-mating intervals remained comparable between groups.

A higher incidence of stillborn pups and associated herewith a lower incidence of live pups was observed at 80 mg/kg bw/day. A lower number of implantations sites was noted at the same dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

80 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No external abnormalities were seen.
A decreased incidence of pups showing a ringtail was present in both the 20 and 80 mg/kg bw/day dosed groups. An increased incidence of pups with bad condition and hypothermia was noted at 80 mg/kg bw/day. This observation is associated with the decreased survival seen in this group.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The survival rate was lower at all doses and at all time intervals.
Based upon the fact that in the present study survival rate in the second generation of this study was comparable between controls and animals dosed at 5 and 20 mg/kg bw/day and that in a previously conducted study this was also the case the decreased survival rate in the first generation of the present study is considered as coincidental.
No adverse effects noted on the survival rate at doses of 5 and 20 mg/kg bw/day in both generations; at 80 mg/kg bw/day survival rate was decreased during lactation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Birth weight, body weight and weight gain comparable between groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

see results described in parental generation P1

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Litter data:
- Litter size: decreased number of live pups at 80 mg/kg bw/day in the first generation
- weight at birth and during lactation: comparable between groups
- survival rate of pups during lactation: decreased survival at 80 mg/kg bw/day
- abnormalities: no teratogenic potential up to 80 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F1

Effect level:

>= 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no teratogenic effects reported

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No external abnormalities were seen.
An increased incidence of bad condition and hypothermia was recorded at 5 mg/kg bw/day but not at 20 and 80 mg/kg bw/day. Therefore, the relevance of these observations is considered coincidental.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Survival rate was, although statistically seen lower, considered to be normal at 5 mg/kg bw/day. This also was the case at 20 mg/kg bw/day. At 80 mg/kg bw:day, survival rate was decreased.

Description (incidence and severity):

Birth weight, body weight and weight gain comparable between groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Litter data:
- litter size: decreased litter size, due to decreased number of implantations, and an increased incidence of stillborn pups at 80 mg/kg bw/day in the second generation
- weight at birth and during laction: comparable between groups
- survival rate of pups during lactation: decreased survival at 80 mg/kg bw/day
- abnormalities: no teratogenic potential up to 80 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F2

Effect level:

>= 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no teratogenic effects reported

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Dosing at 5 and 20 mg/kg bw/day during pre-cohabitation period of 60 days and during mating period and pregnancy during 2 generations did not result in test article related advserse effects. Dosing at 80 mg/kg bw/day resulted in test article related maternal toxicity. Increased presence of waste of food and a descreased bodyweight gain were noted during the pre-cohabitation period, during pregnancy and lactation of both the first and second generation.
histological examination of kidneys, male and female genital tract organs did not reveal primary test article-related changes. in the liver, an increased presence of large vacuoles in the hepatocytes was noted at 80 mg/kg bw/day in the pretreated males of the first generation. The duration of gestation was increased in both the first and second generation further resulting in an increased incidence of dystocia. related to these maternal effects at 80 mg/kg bw, a decreased number of live pups and an increase in number of stillborn pups were present in both generations. The survival rate of pups during lactation was considered to be decreased i both generatoins. No teratogenic potential was found.

Conclusions:

In this two-generation study, parental toxicity was seen at the highest dose (reduced body weight and body weight gain, increased incidence of pilo-erection and, in P1 males, vacuolisation of hepatocytes). At the high dose 80 mg/kg bw/day, a reduced gestation rate and increased duration of gestation were also seen in females, the latter considered responsible for the concurrent increased rate of dystocia. At the highest dose, reproductive toxicity was seen as a slightly reduced number of implantations, reduced number of live pups and offspring survival and increased number of stillborn pups. No teratogenic effects were reported.

Executive summary:

From the above data, it can be concluded that dosing at 5 and 20 mg/kg bw/day during pre-cohabitation period of 60 days and during mating period and pregnancy during 2 generations did not result in test article related adverse effects. Dosing at 80 mg/kg bw/day resulted in test article related maternal toxicity. Increased presence of waste of food and decreased body weight gain were noted during the pre-cohabitation period, during pregnancy and lactation of both the first and the second generation. Histological examination of kidneys, male and female genital tract organs did not reveal primary test article-related changes. In the liver, an increased presence of large vacuoles in the hepatocytes was noted at 80 mg/kg nw/day in the pretreated males of the first generation. The duration of gestation was increased in both the first and second generation further resulting in an increased incidence of dystocia. Related to these maternal effects at 80 mg/kg, a decreased number of live pups and an increase in number of stillborn pups were present in both generations. The survival rate of pups during lactation was considered to be decreased in both generations. No teratogenic potential was found.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

chronic

Species:

rat

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 the rat two-generation study (Dirkx et al., 1992b), fertility was not affected, but a reduction in gestation rate and increases in duration of gestation and rate of dystocia were observed in female animals exposed to the highest dose (80 mg/kg bw/day). At this dose, maternal toxicity was also observed, as indicated by reduced body weight and body weight gain. Furthermore, a slightly reduced number of implantations were observed at this dose. No information is presented in the CLH report to indicate whether these effects were seen in all generations. From the CAR (2009) it appears that the increased gestation duration and dystocia occurred in both generations, whereas the reduced gestation rate occurred in the first generation and the reduced implantations in the second generation. Given the limited information available (on e.g. number of animals affected, magnitude of the effects), it is difficult to judge whether there indeed is an effect and whether there is a causal relationship, as required according to CLP section 3.7.2.3.4. Hence, the RAC was provided with too little study details to allow proper evaluation of the endpoint ‘effects on sexual function and fertility’.

Source: Committee for Risk Assessment RAC. Opinion proposing harmonised classification and labelling at EU level of Imazalil (ISO). CLH-O-0000002720-08-03/F. Adopted 4 June 2013.
Reference: Dirkx, P., Lampo, A., Vandenberghe, J., Coussement, W. & Van Cauteren, H. (1992b) R 23979—imazalil 2-generation reproduction study with 1 litter per generation in Wistar rats. Administration orally through the diet. Unpublished report No 2337 from Department of Toxicology, Janssen Research Foundation, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV.

Effects on developmental toxicity

Description of key information

Developmental toxicity of Imazalil and its salts, including Imazalil sulphate, was evaluated in a two-generation study in rats and four developmental toxicity studies: two in rabbits, one in rats and one in mice.

To cover this endpoint under REACH, 2 of these studies are entered as Robust Study Summaries:

- in the two-generation study in rats with Imazalil (Dirkx et al., 1992b, K2) mentioned above, teratogenic effects were not reported.

- a rat developmental toxicity study with Imazalil sulphate (Gillardin et al., 1998, K2): rats were treated with Imazalil sulphate at the following dose levels: 0-40-80-120 mg/kg bw/d. Treatment with Imazalil sulphate resulted in reduced food consumption as well as body weight or body weight gain during the dosing period at or above the lowest tested dose of 40 mg/kg bw/d in rats. NOAEL for developmental toxicity was 40 mg/kg bw/d in the rat.

Based on the available data there are no indications of teratogenic effects of Imazalil or imazalil sulphate, and the adverse effects observed on fertility or development are associated with maternal toxicity, or occurred at doses not significantly below the maternal LOAEL. Therefore, classification and labelling for reproductive toxicity is not required.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-03-08 to 1988-04-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Janssen Pharmaceutica laboratories; lot ZR027180G1A631. Agroform 047701 was used (Imaezalil 498.6 g/L)
- Appearance: off-white to beige coloured powder
- Expiration date of the lot/batch: no data
- Purity test date: 1998-03-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in closed containers
- Stability under test conditions: known
- Solubility and stability of the test substance in the solvent/vehicle: no data

FORM AS APPLIED IN THE TEST (if different from that of starting material) : given as a solution or a suspension

Species:

rat

Strain:

Sprague-Dawley

Remarks:

OFA.SD. (IOPS Caw)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO , young and healthy virgin female rats
- Age at study initiation: 2-3 months ; sexually mature at the start of the study
- Weight at study initiation: 245.9 - 252.4 g
- Fasting period before study: no
- Housing: plastic cages with 6 females per cage (37x20x15 cm), and isolated as soon as sperm is observed until the end of pregnancy
- Diet (e.g. ad libitum): ad libitum, pelleted rat food, administered in self-raising hoppers
- Water (e.g. ad libitum): ad libitum, administered in drinking bottles
- Acclimation period: at least one week under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +- 2°C
- Humidity (%): 50 +- 20 %
- Air changes (per hr): 9
- Photoperiod (hrs dark / hrs light): 12h/day

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article i given orally by gavage as a solution or a suspension and 1mL per 100g body weight is given.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 1ml/100 g bw
- Lot/batch no. (if required): not specified
- Purity: not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1F/2M
- Length of cohabitation: until sperm is observed
- No replacement of first male by another male with proven fertility after number of days of unsuccessful pairing
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy
- Any other deviations from standard protocol: males are used for breeding and are not dosed

Duration of treatment / exposure:

from day 6 to day 16 of the pregnancy

Frequency of treatment:

daily

Duration of test:

22 days

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

120 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

No. of animals per sex per dose:

24 females per group, 4 groups

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Dose levels are determined following review of available data in pharmacology, kinetics and toxicity studies. Dose determination aims to include a surely toxic dose and a no-effect dose level.
- Rationale for animal assignment (if not random): random
- Justification for selection of the test system: The rat is selected following review of available data on toxicity, pharmacology and/or pharmacokinetics and also because of the possibility to compare the hereby recorded data with the accumulated control data of rats.
- Route of administration: this route of administration is chosen following review of available data on pharmacology and/or because this route is the intended route for therapeutic applications of the test article in man and/or animal.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations include examination of signs of waning health, abnormal behaviour or unusual appearance, occurence of untoward clinical effects and manifestations of toxic and pharmacological response, morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: in the morning of days 1, 6, 17 and 22 of the presumed pregnancy. Body weights are also determined daily during the dosing period (day 6 through day 16) for adjustment of dose.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Individual records are made prior to, during and after dosing or respectively in the morning of day 6 (non-dosing period: day 1 through day 5), in the morning of day 17 (dosing period: day 6 through day 16) and in the morning of day 22 (non dosing period : day 17 through day 21).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Prior to sacrifice, a complete physical examination is performed on all surviving rats
- Sacrifice on gestation day 22: all surviving females by decapitation
- autopsy is performed as soon as possible after death during the stud or after sacrifice at the end of the study and any macroscopic pathological changes are noted

ORGAN WEIGHT:
- after removing the uterus in toto, the weight of the uterus is recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included: Gravid uterus weight, Number of corpora lutea, Number of implantations, Number of resorptions, Number of live and dead fetuses, any abnormal condition
- in case a reduced litter size (0 to 3 fetuses) is noted, the Salewski technique is performed in order to discriminate between resorption and pseudopregnancy

Fetal examinations:

- External examinations: Yes
- all per litter
- all live fetuses are individually weighed
- all live and dead fetuses are carefully examined for any external anomalies
- Soft tissue examinations: No
- Skeletal examinations: Radiographic examinations are carried out for all fetuses of all groups. Rat fetuses of each litter are randomized for dissection (half) and if indicated by the results of the radiographic examination for clearing and bone staining with alizarin (half).
- Head examinations: No
- Malformations or fetal anomalies encountered at the moment delivery by caeserean section or at each stage of dissection are noted

Statistics:

Adult rat:
- mortality and pregnancy: Chi-square test
- body weight, bodyweight change and food consumption: Mann-Whitney U test

Litter data:
live fetuses, dead fetuses, resorbed fetuses, litter size, body weight, abnormalities: Mann-Whitney U test

Indices:

no data

Historical control data:

no data

Clinical signs:

not specified

Description (incidence and severity):

No data on clinical signs is available

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (n° 34-not pregnant of the 40 mg/kg dosed group) died for unknown reason on day 12. This animal did not show drug- or dose-related effects.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was an increase in weight among all dams of control as well as dams doses at 40, 80 and 120 mg/kg. Lower body weight was observed at 40, 80 and 120 mg/kg at the end of dosing period (day 17) and at 120 mg/kg at the end of post-dosing perdiod. In addition, body weight change was lower when compared to controls in the 80 and 120 mg/kg.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption significantly decreased during dosing in the three dosed groups compared to controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Description (incidence and severity):

No further data available

Gross pathological findings:

not specified

Description (incidence and severity):

No further data available

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No significant difference wasnoticed in the number of implantations between the various groups.

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 80 mg/kg resorption rate showed a tendency for increase (statistically not significant). At 120 mg/kg bw, this increase was statistically significant (p<0.01). At 120 mg/kg this finding resulted in a lower litter size (p<0.01) and a decreased number of live fetuses (p<0.01).

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

At 120 mg/kg there was a decreased number of live fetuses (p<0.01)

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Althought somewhat fluctuating between groups, pregnancy rate was comparable between groups:
Control group: 24/24 or 100 %
40 mg/kg: 20/24 or 83%
80 mg/kg: 23/24 or 96%
120 mg/kg: 22/24 or 92%

Other effects:

not specified

Key result

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean weight was comparable between controls and the 40 mg/kg dosed group. At 80 and 120 mg/kg body weight of pups decreased (p<0.05 and p<0.001, respectively).

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Decreased number of live fetuses observed at 120 mg/kg bw.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Fetal sex ratios determined for the dosed groups were 0.84, 0.96 and 0.91 respectively whereas in the controls the ratio was 0.76 which is slightly lower value than normally expected.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Lower litter size at 120 mg/kg bw.

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Split vertebrae center, rudimentary 14th ribs and wavy ribs are skeletal variations revealed by the X-ray method and confirmed by the alizarin staining. These findings are reguarly encountered in rat fetuses.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Coelosomia and macroglossia are visceral abnormalities that might occur spontaneously and are randomly distributed among control and dosed groups.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

It is concluded that no teratogenic effects were evidenced in this study.

Key result

Dose descriptor:

LOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Litter data

			Fetuses (number expressed as mean+/-SE)
dosage groups	litter size	N	live	dead	resorbed
control group	13.9	24	13.8 +/-0.8	0.0	0.4 +/-0.1
40 mg/kg	12.1	20	12.0 +/-1.1	0.1 +/-0.1	0.5 +/-0.2
80 mg/kg	13.0	23	13.0 +/-0.9	0.0	2.0 +/-0.7
120 mg/kg	11.2**	22	11.1 +/-0.9**	0.1 +/-0.1	3.7 +/-0.7***

N: the number of litters examined per group

**p<0.01; ***p<0.001

Body weight

Dosage groups	N	weight of pups (grams)
Control	24	5.6 +/-0.1
40 mg/kg	20	5.5 +/-0.1
80 mg/kg	22	5.2 +/-0.1*
120 mg/kg	22	4.6 +/-0.1***

N represents the number of litters examined per group

*p<0.05; ***p<0.001

Conclusions:

The test substance, when administered orally to pregnant Sprague-Dawley rats from day 6 through day 16 of pregnancy at the dose of 40 mg/kg was slightly maternally toxic (decreased food consumption and body weight at the end of the dosing) but without any adverse effect on litter parameters. Dosing at 80 and 120 mg/kg also resulted in maternal toxicity (decreased food consumption, body weight at the end of dosing and body weight gain) and herwith associated, in slight to moderate embryonal resorption and decreased pup weight. No teratogenic effect was evidenced up to the dose of 120 mg/kg in rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subchronic

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The developmental toxicity studies in rats (Gillardin et al., 1988) and rabbits (Dirkx, 1992a) were conducted with Imazalil sulphate, whereas in another developmental toxicity study in rabbits that showed no effects (Dirkx and Marsboom, 1985) Imazalil nitrate was administered. The read-across from these salts to Imazalil is considered acceptable because of the good water solubility of both substances. The opposite is also considered: the read-across from Imazalil (and Imazalil nitrate) to Imazalil sulphate is considered acceptable.

The table below presents the available information on the toxicity to reproduction of Imazalil and its salts:

	Dirkx & Marsboom, 1985	Dirkx, et al., 1992a	Dirkx, et al., 1992b	Dirkx, et al., 1992c	Gillardin et al., 1988
Test item	Imazalil nitrate	Imazalil sulphate	Imazalil	Imazalil sulphate	Imazalil sulphate
Species	rabbit	rabbit	rat	mouse	rat
Exposure duration	day 6 to 18 of gestation	day 6 to 18 of gestation	two-generation study	day 6 to 16 of gestation	day 6 to 16 of gestation
Guideline	similar OECD 414	similar OECD 414	similar OECD 416	similar OECD 414	similar OECD 414
Route of exposure	oral	oral (gavage)	oral (diet)	oral (gavage)	oral (gavage)
Dose tested	0-1-2.1-4.1 mg/kg bw/d imazalil equivalents (imazalil nitrate)	0-5-10-20 mg/kg bw/d imazalil equivalents (imazalil sulphate)	0-5-20-80 mg/kg bw/d	0-10-40-80-120 mg/kg bw/d imazalil equivalents (imazalil sulphate)	0-40-80-120 mg/kg bw/d
Entered in IUCLID	/	/	section 7.8.1	section 7.8.2 (supporting study)	section 7.8.2 (key study)
Reference from the RAC Opinion	yes	yes	yes	no	yes

The information summarized below is extracted from the RAC opinion on harmonised classification and labelling of Imazalil, and complemented with information from additional sources.

• In the rat two-generation study (Dirkx et al., 1992b, K2), an increased number of stillborn pups, a decreased number of live pups and a reduced pup survival were observed at a dose at which also parental toxicity was seen (80 mg/kg bw/d). From the CAR (2009) it appears that these effects occurred in both generations, but this information was not presented in the CLH report.

• In the developmental toxicity study with rats (Gillardin et al., 1988, K2), pup weight was reduced at the mid and high dose (80 and 120 mg/kg bw/d), and the high dose also resulted in a reduced number of live foetuses and increased resorptions. However, maternal toxicity (as evidenced by reduced bw and food consumption during dosing) was already observed at the lowest dose tested of 40 mg/kg bw/d.

• Similar effects were observed in the developmental toxicity study with rabbits (Dirkx, 1992a) but here the high dose (20 mg/kg bw/d) also caused increased mortality (8 out of 15 dams). Effects on the offspring at the mid and high dose (10 and 20 mg/kg bw/d) included reduced litter-size, reduced number of live foetuses, and an increased number of post-implantation losses. The effects were dose-related but not statistically significant, and occurred in the presence of maternal toxicity (reduced bw and food consumption).

• The developmental toxicity study in rabbits showed no effects (Dirkx and Marsboom, 1985) after administration of Imazalil nitrate.

• In the developmental toxicity study in mice (Dirkx et al., 1992c), no adverse effects were noted at 10 mg/kg. Dosing at 40 mg/kg resulted in slight maternal toxicity (one mortality, slightly decreased food consumption and uterus weight) and slight fetal toxicity (tendency to increased resorptions; tendency to decreased litter size and number of live fetuses, although ratios were comparable when the predosing number of implantations was taken into account). At 80 and 120 mg/kg, maternal and fetal toxicity was more prominent. Maternal toxicity was characterized by mortality (1 and 2 mortalities), decreased body weight and food consumption and decreased uterus weight. Related to this maternal toxicity, a decreased number of live fetuses (resulting in a smaller litter size), an increase in resorptions and an increased incidence of some minor skeletal variations were observed at 80 and more prominently at 120 mg/kg bw. No teratogenic potential was observed. It is therefore considered that 10 mg/kg bw/day in mice is a maternal and developmental OEL, whereas slight toxicity was present at 40 mg/kg. The doses of 80 and 120 mg/kg were moderately toxic dose levels.

Imazalil treatment did not result in malformations in either rats or rabbits, but in both species Imazalil induced an increase in resorptions and a reduction in live foetuses at dose levels also inducing maternal toxicity. The available data for rats are too limited (no data on magnitude of the effects) to allow a proper assessment. From the developmental toxicity study in rabbits somewhat more (but still limited) information is available. The foetal effects observed at 20 mg/kg bw/d in the rabbit study are not considered relevant for classification, given the excessive mortality rate (53%) in dams. The maternal toxicity at 10 mg/kg bw/d is not considered to be excessive. It can however not be assessed with the limited data available (no information on e.g. net weight gain) whether the reduced bw and food consumption were a primary effect or secondary to the post-implantation loss. Overall, the RAC was provided with too little study details to allow proper evaluation of the endpoint ‘developmental toxicity’.

The RAC noted that EFSA in their peer review of Imazalil (2010) concluded that Imazalil is not a reproductive toxicant or a teratogen. RAC, however, did not find the information provided detailed enough to evaluate this hazard class and hence no conclusion on reproductive toxicity was agreed.

Source: Committee for Risk Assessment RAC. Opinion proposing harmonised classification and labelling at EU level of Imazalil (ISO). CLH-O-0000002720-08-03/F. Adopted 4 June 2013.

References:

• Dirkx, P. & Marsboom, R. (1985) R 18531 - Imazalil nitrate. Oral embryotoxicity and teratogenicity study in New Zealand white rabbits (segment II) administration: oral. Unpublished report 1482 (85.18.04). Department of Toxicology, Janssen Pharmaceutica NV, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV.

• Dirkx, P., Lampo, A., Coussement, W. & Van Cauteren, H. (1992a) R 23979—imazalil sulphate. Embryotoxicity and teratogenicity study in albino rabbits. Administration: orally by gavage. Unpublished report No. 2615 from Department of Toxicology, Janssen Research Foundation, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV.

• Dirkx, P., Lampo, A., Vandenberghe, J., Coussement, W. & Van Cauteren, H. (1992b) R 23979—imazalil 2-generation reproduction study with 1 litter per generation in Wistar rats. Administration orally through the diet. Unpublished report No 2337 from Department of Toxicology, Janssen Research Foundation, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV.

• Dirkx, P., Lampo, A., Van Deun, K., Coussement, W. & Van Cauteren, H. (1992c) R 27180: Imazalil sulfate. Embryotoxicity and teratogenicity study in albino mice (segment II). Administration: orally by gavage. Unpublished report No. 2712 from Department of Toxicology, Janssen Research Foundation, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV

• European Food Safety Authority; Conclusion on the peer review of the pesticide risk assessment of the active substance imazalil. EFSA Journal 2010, 8(3):1526. [69 pp.]. doi:10.2903/j.efsa.2010.1526. Available online: www.efsa.europa.eu

• Gillardin, J.M., van Cauteren, H., Sanz, G. & Marsboom, R. (1988) R 27180 embryotoxicity and teratogenicity study in Sprague-Dawley rats (segment II): oral by gavage. Unpublished report No. 2003/88-05 from Research Department, Laboratoires Janssen, Aubervilliers, France. Submitted to WHO by Janssen Pharmaceutica NV.

Justification for classification or non-classification

Based on the available data there are no indications of teratogenic effects of imazalil sulphate, and the adverse effects observed on fertility or development are associated with maternal toxicity, or occurred at doses not significantly below the maternal LOAEL. Therefore, classification and labelling for reproductive toxicity is not required because of a lack of detailed information to agree on a conclusion on reproductive toxicity.