Alcohols, C20-22, reaction products with phosphorus oxide (P2O5)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL reprotoxicity in rat = 1000 mg/kg bw (oral)

Link to relevant study records

Reference

Reference 1

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:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 April 2008 - 11 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

except during study days -1 to 16, and except for the absence of chemical analyses of dosage forms

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder
- Age at first treatment: approx. 10 weeks
- Weight at first treatment (mean): M=408 g, F=269 g
- Housing: individually, except during pairing
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Relative humidity: 50 ± 20%
- Light/dark cycle: 12h/12h
- Ventilation: 12 cycles/hour of filtered, non-recycled air.

IN-LIFE DATES: beginning: 22 April 2008 / end: up to 11 June 2008

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
test item was ground, heated to 80°C, mixed with vehicle heated to 80°C, forming a suspension.
The test item dosage forms were prepared daily

VEHICLE
- Justification for use and choice of vehicle (if other than water): lipophilicity of the substance
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day
- Lot/batch no. (if required): 057K6093
- Purity: not indicated

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred
- Proof of pregnancy: vaginal plug, or sperm in vaginal smear - referred to as day 0 post-coitum
- In case of unsuccessful pairing: not detailed (pairing always succeeded)
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Absence of a practical method of analysis

Duration of treatment / exposure:

from 2 weeks before mating until the end of mating (males: total of 39 days) or day 5 pp (females: total of 43-51 days)

Frequency of treatment:

once daily

Details on study schedule:

- no F1 parents (only one generation mated).
- Age at mating of the mated animals in the study: 13-16 weeks

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day (m/f)
Basis:
other: nominal per gavage

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no serious effects at up to 1000 mg/kg/day in a 14-day range-finding study, see 7.5.1
- Rationale for animal assignment: computerized stratification procedure (average body weight of each group is similar)

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule: day 1 and then weekly; and in F also days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum

FOOD CONSUMPTION for each animal: Yes
- Time schedule: weekly (last 7-day consumption period)

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Yes : from a fresh vaginal lavage, each morning during the mating period, until the females were mated

Sperm parameters (parental animals):

No seminology examinations.
Testis and epididymis : weight (all males), microscopic exmaination (some rats: see table 1).

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight on days 1 and 5, clinical signs

Postmortem examinations (parental animals):

SACRIFICE
- All male survivors: after the end of the mating period
- All female survivors: on day 6 post-partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- Sacrifice on non-selected breeders/progeny: not applicable (only 1 generation of parents and of offspring)
- All pups sacrificed on day 5 post-partum

GROSS NECROPSY: Yes, on pups sacrificed and found dead
- external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS: Not performed

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Mating index = 100 * Number of mated animals / Number of paired animals
Fertility index = 100 * Number of pregnant female partners / Number of mated pairs
Gestation index = 100 * Number of females with live born pups / Number of pregnant females
Live birth index = 100 * Number of live born pups / Number of delivered pups

Offspring viability indices:

Viability index = 100 * Number of surviving pups on day 5 post-partum / Number of live born pups

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY
There were no treatment-related deaths or sacrifices (deaths were due to gavage errors, cranial hematoma or non-pregnancy).
Hypersalivation and reflux at dosing were observed in all groups and may be related to the vehicle and/or the test item but are non-adverse

BODY WEIGHT GAIN:
All female dose-groups gained less body weight than controls during the premating period, but this did not continue during gestation and lactation and was therefore considered to be non-adverse.

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: NOAEL = highest tested dose. All female dose-groups gained less body weight than controls during the premating period, but this did not continue during gestation and lactation and was therefore considered to be non-adverse.

Dose descriptor:

NOEL

Remarks:

reproduction (mating and fertility)

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: NOEL = highest tested dose, without relevant effects.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Not required (no effects)

Dose descriptor:

NOEL

Remarks:

offspring toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: NOEL = highest tested dose, without relevant effects.

Reproductive effects observed:

not specified

Not required (no effects)

Conclusions:

Under on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day, the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day and the NOEL for toxic effects on progeny was 1000 mg/kg/day.

Executive summary:

The test item, LCE07103, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until day 5 post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

There were no treatment-related deaths during the study and the only clinical signs observed were hypersalivation and reflux at dosing, which were considered to be related to treatment with the test item but are non-adverse. All female groups treated with LCE07103 gained less body weight during the premating period but this did not continue during gestation and lactation and was therefore considered to be non-adverse. There were no effects at any dose-level on food consumption. There were no differences from controls for pairing, mating and fertility parameters. Pups showed no effects of treatment on survival or body weight performance. No treatment-related macroscopic or microscopic findings were noted and there were no treatment-related organ weight changes.

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

without detailed documentation

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S5(R2) Detection of toxicity to reproduction for medicinal products and toxicity to male fertility

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

(no postnatal observations of pups)

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: (P) males 6-7 wks, females 10-11 weeks
- Weight at study initiation: (P) males 193-240 g; females 208-262 g
- Fasting period before study: no
- Housing: according to the investigators "during the acclimation and premating periods, 10 rats (5 males and 5 females) were housed per TR18 stainless-steel cage..."; during mating, 1 male and 1 female housed in RB3-modified high-grade polypropylene cage with stainless-steel mesh lids and floors; during gestation, 5 females per RB3-modified cage; after mating, 5 males per TR18 cage
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): expanded rodent diet (Special Diet Services Ltd.), ad libitum
- Water (e.g. ad libitum): public supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18
- Humidity (%): 55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

other: 1% w/w aqueous Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- test material weighed into glass container and heated to ~80 deg C until molten
- vehicle heated to 75 deg C
- test material and vehicle combined using continuous magnetic stirring, 20% behenyl alcohol
- suspension cooled slowly to <60 deg C
- further cooled to 30 deg C
- slowly homogenized <=2 min
- cooled to room temperature
- 20% suspension prepared weekly
- 20% suspension provided top dose
- mid and low dose prepared on day of use by dilution with vehicle; 20% suspension magnetically stirred prior to removal of aliquots for dilution; dilutions hand swirled prior to magnetic stirring

VEHICLE
- Justification for use and choice of vehicle (if other than water): not stated
- Concentration in vehicle: 20, 2 and 0.2%
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): no data
- Purity: 1% aqueous

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: not stated
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After ... days of 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): 5/cage; RB3-modified cages
- Any other deviations from standard protocol: OECD guideline 415 recommends that: pregnant females are house individually, the mating period should be 3 weeks

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Males: from 71 days prior to mating, during mating and until females sacrificed
Females: from 15 days prior to mating, during mating, and up to day 17 of gestation; killed on day 20 of gestation

Frequency of treatment:

daily

Details on study schedule:

1-generation study

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: previous repeated dose toxicity study NOAEL was 1000 mg/kg bw/day
- Rationale for animal assignment (if not random): no data

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: evidence of reaction to treatment, moribund condition, mortality

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: males and females twice weekly prior to mating; males twice weekly after mating; females on gestation days 0, 3, 7, 10, 14, 18 and 20

FOOD CONSUMPTION :
- Food consumption: Yes
- Time schedule: males weekly prior to mating, females daily prior to mating, females on gestation days 0-2, 3-6, 7-9, 10-13, 14-17, 18-19

WATER CONSUMPTION: Yes
- Time schedule: males weekly prior to mating, females daily prior to mating, females on gestation days 0-2, 3-6, 7-9, 10-13, 14-17, 18-19

Oestrous cyclicity (parental animals):

10 days prior to mating, daily vaginal smears to assess regularity and duration of oestrus cycles

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight, sperm count in epididymides, sperm motility

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, F1 generation examined as foetuses on day 20 of gestation

PARAMETERS EXAMINED
The following parameters were examined in parental females and F1 offspring: numbers of implantation sites, early and late resorptions and viable foetuses; distribution of foetuses in each uterine horn; placental weight; macroscopic examination of placentae; number and sex of foetuses

EXAMINATION OF PUPS: yes, for external and internal abnormalities (visceral and skeletal)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following necropsy of the females
- Maternal animals: All surviving animals on day 20 of gestation

GROSS NECROPSY
- Gross necropsy of females consisted of reproductive endpoints only
- Gross necropsy of males consisited of macroscopic examination externally and internally; sperm assessment

HISTOPATHOLOGY / ORGAN WEIGHTS
No tissues were prepared for microscopic examination
Reproductive organs were weighed

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were examined on day 20 of gestation
- These animals were subjected to examination as follows: each foetus weighed; detailed external examination; contents of cervical, thoracic and abdominal cavities removed from half the foetuses and examined and sex recorded; these foetuses stained for skeletal examination; remaining foetuses examined for visceral abnormalities

HISTOPATHOLOGY / ORGAN WEIGTHS
No tissues prepared for microscopic examination or weighed.

Statistics:

One-way analysis of variance, t-tests - body weight, body weight change, food and water consumption; Dunnetts' or Behren's-Fisher's tests - organ weights; nested analysis of variance, weighted t-tests - foetal and placental weights

Reproductive indices:

number of pregnant females, fertility

Offspring viability indices:

number of viable young (offspring evaluated as foetuses on day 20 of gestation)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- females, no mortality
- males, one death in top dose group in week 6, not considered to be treatment related in the absence of toxic signs in any other animals

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- no effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- no effects

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- PREGNANCY RATE - no effects (22, 22, 22 and 21 in control, low, mid and high dose groups respectively)
- no effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- no effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
- males, reproductive organs, no effects

GROSS PATHOLOGY (PARENTAL ANIMALS)
- no effects

HISTOPATHOLOGY (PARENTAL ANIMALS)
- not examined

OTHER
- REPRODUCTIVE PARAMETERS
- Number of corpora lutea - no effects (17.8, 18.4, 18.7 and 18.9 for controls, low, mid and high dose respectively)
- Number of implantations - no effects (means 17.2, 17.0, 18.1 and 18.0 for controls, low, mid and high dose respectively)
- Number of viable young - no effects (means 16.4, 15.9, 17.0 and 16.9 for controls, low, mid and high dose  respectively)
- Sex ratio - no effects
- Number of resorptions (early or late) - no effects
- Pre-implantation loss - no effects (3.3, 8.3, 3.2,  5.8% for controls, low, mid and high dose respectively)
- Post-implantation loss - no effects (4.7, 6.4, 6.3 and 5.8% for controls, low, mid and high dose respectively)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
- no effects

CLINICAL SIGNS (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

BODY WEIGHT (OFFSPRING)
- no effects

SEXUAL MATURATION (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

ORGAN WEIGHTS (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

GROSS PATHOLOGY (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

HISTOPATHOLOGY (OFFSPRING)
- not applicable, foetuses examined at day 20 of gestation

OTHER FINDINGS (OFFSPRING)
- MACROSCOPIC EXAMINATION (OFFSPRING)
- no variations were observed that were not comparable to historical control values
- SKELETAL EXAMINATION (OFFSPRING)
- no variations were observed that were not comparable to historical control values
- VISCERAL EXAMINATION (OFFSPRING)
- no variations were observed that were not comparable to historical control values

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Reproductive effects observed:

no

Conclusions:

In a reliable study, conducted to a protocol similar to OECD guideline 415, an NOAEL of 1000 mg/kg bw/day was determined in the rat for reproductive effects. The study was performed in compliance with GLP.

Executive summary:

[In view of the structural and chemical similarities, it is considered that the results of this study can be used for read-across to C12, 13, 14 and 15 alcohols; linear and monobranched.]

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well-conducted study which meets basic scientific principles. The study is a read across from 1-docosanol (CAS 661-19-8).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline required

Principles of method if other than guideline:

Repeated dose toxicity test in which rats were orally dosed daily for 26 weeks and reproductive organs assessed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: ~21-28 days at purchase
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: 5/cage in stainless steel cages, containing absorbent paper
- Diet (e.g. ad libitum): expanded rodent diet (Special Diets Services, UK), ad libitum
- Water (e.g. ad libitum): public supply (Suffolk Water Company, UK), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- test material heated to approx. 80 deg C
- vehicle heated to approx. 75 deg C
- vehicle added to test material while being magnetically stirred at high speed
- resulting 20% (w/w) suspension homogenized and slowly cooled to below 60 deg C
- when cooled to 30 deg C, suspension slowly homogenized again for >=2 min
- cooled to room temp.
- stored at 13 deg C
- prepared once weekly
- 20% suspension used for top dose; for low and mid doses, suspension magnetically stirred and aliquots taken for dilution on day of use; constant dose volume of 5 ml/kg bw per dose
- dilutions mixed by hand swirling followed by magnetic stirring

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 1%
- Amount of vehicle (if gavage): 5 ml/kg bw per dose
- Lot/batch no. (if required): no data
- Purity: no data

Details on mating procedure:

No mating - screening test

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

No mating - screening test

Remarks:

Doses / Concentrations:
10, 100, 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random
- Other:
- Repeated dose toxicity (oral) study - acceptable as reproductive screen since reproductive organs were included in those evaluated

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS (including mortality): Yes
- Time schedule: >=twice daily
- Cage side observations included: evidence of reaction to treatment or moribund condition, evidence of ill health such as blood or loose faeces

DETAILED CLINICAL OBSERVATIONS: Yes, individual observations
- Time schedule: once daily during week 1, twice weekly during weeks 2 to 4, once weekly during weeks 5 to 13, once every 2 weeks from week 14 onwards

BODY WEIGHT: Yes
- Time schedule for examinations: pre-study, weekly during the study or more frequently if appropriate (for animals in moribund condition), at necropsy

FOOD CONSUMPTION:
- Food consumption for each cage determined: Yes

FOOD EFFICIENCY:
- Weekly group mean food conversion efficiencies calculated from the consumption and body weight gain data: Yes, for the first 14 weeks of treatment

WATER CONSUMPTION: No

OTHER: Ophthalmoscopic examination, haematology, clinical chemistry, urinalysis - reported elsewhere

Oestrous cyclicity (parental animals):

not examined; ovaries and uterus (with cervix) weighed and examined

Sperm parameters (parental animals):

not examined; testes and epididymides weighed and examined

Litter observations:

no litters - not mated - screening test

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: after 26 weeks of treatment
- Maternal animals: after 26 weeks of treatment

GROSS NECROPSY
- Yes

ORGAN WEIGHTS: adrenals, brain, kidneys, liver, lungs (with main stem bronchi), ovaries, pituitary, prostate, spleen, testes, thymus, thyroid (with parathyroids), uterus, cervix

HISTOPATHOLOGY: Yes - adrenals, brain, eyes, optic nerve, femur, heart, kidneys, liver, lungs, seminal vesicles, spinal cord, stomach, thyroid, uterus

Postmortem examinations (offspring):

no offspring - not mated - screening test

Statistics:

Bartlett's test for homogeneity of variance (organ weights, body weight changes); if significant, Behrens-Fisher test, otherwise Dunnett's test.
Two-tailed Fisher's exact test (macroscopic/microscopic pathological findings).

Reproductive indices:

not mated - screening test

Offspring viability indices:

no offspring - not mated - screening test

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

CLINICAL SIGNS AND MORTALITY
- one male in the mid-dose group died at week 25 (examination suggested aspiration of test material through mis-dosing; not considered to be treatment-related)
- no other clinical signs of systemic toxicity or mortality

BODY WEIGHT AND WEIGHT GAIN
- no effects

FOOD CONSUMPTION
- presumably no effects

FOOD EFFICIENCY
- no effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- not examined - screening test

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- not examined - screening test

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- not examined - screening test

ORGAN WEIGHTS (PARENTAL ANIMALS), including testis, epididymis, ovaries, uterus (with cervix)
- no effects

GROSS PATHOLOGY (PARENTAL ANIMALS), including testis, epididymis, ovaries, uterus (with cervix)
- no effects

HISTOPATHOLOGY (PARENTAL ANIMALS), including testis, epididymis, ovaries, uterus (with cervix)
- no effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

other: Generation: not mated - screening study

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

no offspring - not mated - screening test

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Conclusions:

In a reliable screening study, a repeated oral dose NOAEL of 1000 mg/kg bw/day was determined for effects on reproductive organs in the rat. The result is a read across from 1-docosanol (CAS 661-19-8).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The reproductive toxicity of C20-22 alkyl phosphate was evaluated in rats by oral route in one subacute toxicity study performed according to OECD 422 and in compliance with GLP. This study was scored as validity 1 according to Klimisch criteria and therefore was selected as the Key study. The results of this study were supported and completed by those observed in an one-generation study with docosan-1-ol performed according to ICH guideline (performed by oral route in the rats) and a repeated-dose toxicity study with docosan-1-ol.

OECD TG 422 with C20-22 alkyl phosphate (rat, oral):

C20-22 alkyl phosphate was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until day 5 post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

There were no treatment-related deaths during the study and the only clinical signs observed were hypersalivation and reflux at dosing, which were considered to be related to treatment with the test item but are non-adverse. All female groups treated with C20-22 alkyl phosphate gained less body weight during the premating period but this did not continue during gestation and lactation and was therefore considered to be non-adverse. There were no effects at any dose-level on food consumption. There were no differences from controls for pairing, mating and fertility parameters. Pups showed no effects of treatment on survival or body weight performance. No treatment-related macroscopic or microscopic findings were noted and there were no treatment-related organ weight changes.

Under on the experimental conditions of this study:

-       the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day,

-       the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day, and

-       the NOEL for toxic effects on progeny was 1000 mg/kg/day.

One-generation study with docosan-1-ol (rat, oral):

Docosan-1-ol was administered daily by oral gavage to male and female Sprague-Dawley rats, at dose-levels of 10, 100 or 1000 mg/kg/day. Males were treated from 71 days prior to mating, during mating and until females sacrificed, and females from 15 days prior to mating, during mating, and up to day 17 of gestation (killed on day 20 of gestation).

In this study a NOAEL of 1000 mg/kg bw/day was determined in the rat for reproductive effects.

Repeated-dose toxicity study with docosan-1-ol (rat, oral):

Docosan-1-ol (CAS 661-19-8) was administered daily to groups of rats at levels up to 1000 mg/kg for 26 weeks. Body weight and food consumption was not affected by treatment. Haematology, clinical chemistry and gross necropsy investigations showed no evidence of toxicity. There were no treatment related microscopic changes.

a repeated oral dose NOAEL of 1000 mg/kg bw/day was determined for effects on reproductive organs in the rat.

Effects on developmental toxicity

Description of key information

NOAEL maternal = or > 361 mg/kg bw/day (no significant effect observed)

NOEL (offspring) = or > 361 mg/kg bw/day (no significant effect observed)

(based on related substances)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

without detailed documentation

Qualifier:

according to guideline

Guideline:

other: ICH Harmonized Tripartite Guideline S5 (R2) for Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

(non standard examination of soft tissue and head of foetuses)

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Froxfield SPF Rabbits Ltd., UK
- Age at study initiation: 18-26 weeks on arrival
- Weight at study initiation: 3.29-4.98 kg at start of study
- Fasting period before study: no data
- Housing: individually in suspended stainless-steel cages (TR6)
- Diet (e.g. ad libitum): standard rabbit diet (Special Diets Services Ltd., UK), ad libitum
- Water (e.g. ad libitum): public supply, ad libitum
- Acclimation period: >=1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18
- Humidity (%): 55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

other: 1% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- test material weighed into glass container and heated to ~80 deg C until molten
- vehicle heated to 75 deg C
- test material and vehicle combined using coninuous magnetic stirring, 20% behenyl alcohol
- suspension cooled slowly to <60 deg C
- further cooled to 30 deg C
- slowly homogenized <=2 min
- cooled to room temperature
- 20% suspension prepared weekly
- 20% suspension provided top dose
- mid and low dose prepared on day of use by dilution with vehicle; 20% suspension magnetically stirred prior to removal of aliquots for dilution; dilutions hand swirled prior to magnetic stirring

VEHICLE
- Justification for use and choice of vehicle (if other than water): not stated
- Concentration in vehicle: 20, 2 and 0.2%
- Amount of vehicle (if gavage): 10 ml/kg bw for vehicle control and top dose groups; 0.625 and 2.5 ml/kg bw for low and mid dose groups respectively
- Lot/batch no. (if required): no data
- Purity: 1%

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused with males of establised fertility
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: no data
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: not specified, but referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no data

Duration of treatment / exposure:

days 6-19 of gestation

Frequency of treatment:

daily

Duration of test:

females killed on day 29 of gestation

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: 28 days
- Dose selection rationale: based on previous range-finding study
- Rationale for animal assignment (if not random): randomly allocated to the four treatment groups in order of mating "to evenly distribute the mated females among the groups"
- Other:
- approximately 2 weeks prior to arrival of females at testing facility, oestrus synchronised by supplier by intravenous injection of 25 IU luteinizing hormone
- following insemination, females injected intravenously with 25 IU luteinizing hormone to ensure successful ovulation
- examined on day 6 of gestation, prior to dosing, to determine suitability for use in study

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: evidence of reaction to treatment or moribund condition

DETAILED CLINICAL OBSERVATIONS: no data

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time schedule for examinations: days 1-5, days 6-12, days 13-19, days 20-23, days 24-28

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: yes, macroscopic examination
- Sacrifice on gestation day 29
- Organs examined in addition to uterine contents and ovaries: no data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- Number of viable young: males, females and total
- Distribution of foetusus in each uterine horn
- Uterus of any female presumed non-pregnant stained and examined for implantation sites

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter - cervical, thoracic and abdominal cavities dissected and contents examined microscopically
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: one third per litter
- Other: all per litter
- foetal body weight
- position of foetus in uterus
- placental weight

Statistics:

One-way analysis of variance, t-tests - body weight, body weight change, food and water consumption; Dunnett's or Behren's-Fisher's tests - organ weights; nested analysis of variance, weighted t-tests - foetal and placental weights

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
no effects other than pale faeces in animals of the top dose group

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
no effects

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

no

Table 1: Reproductive and developmental parameters

Observation	Dose (mg/kg bw/day)
	0	125	500	2000
Animals Assigned (Mated)	22	22	22	22
Animals Pregnant Pregnancy Rate (%)^a	20 91%	19 86%	19 86%	20 91%
Nonpregnant	2	3	3	2
Total litter loss (%)^a	1 10.0%	1 5.3%	1 5.3%	0 0.0%
Corpora Lutea/Dam (mean±SD)	12.8±3.1	12.9±2.2	12.6±3.0	12.2±3.9
Implantations/Dam (mean±SD)	11.4±3.9	11.1±2.6	11.0±3.3	10.6±4.3
Live Fetuses/Dam (mean±SD) Male (mean±SD) Female (mean±SD)	10.1±3.7 4.6±2.6 5.5±2.4	9.8±2.1 4.8±1.5 4.9±1.7	9.3^b±2.6 3.8±1.5 5.5±2.1	9.0±3.8 4.7±2.2 4.3±2.5
Resorptions/Dam (mean±SD) Early (mean±SD) Late (mean±SD)	1.4±1.2 0.4±0.6 1.0±1.0	1.3±1.2 0.3±0.5 1.1±1.0	1.7±1.3 0.4±0.6 1.2±1.1	1.6±1.2 0.7±0.8 0.9±0.9
Preimplantation Loss (%)	10.4	14.2	13.9	13.5
Postimplantation Loss (%)	12.1	12.0	15.2	14.7

^aCalculated for this table

^bIncludes one foetus not sexed at necropsy

Conclusions:

In a reliable study, conducted according to a protocol similar to OECD guideline 414, the NOAEL for maternal toxicity, teratogenicity and foetotoxicity in rabbits, was 2000 mg/kg/day (highest dose tested). The study was performed in compliance with GLP.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

without detailed documentation

Principles of method if other than guideline:

Method: other

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: gavage

Duration of treatment / exposure:

For 15 days prior to mating, during mating and up to Day 17 of gestation.

Frequency of treatment:

daily

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Control animals:

yes

Details on study design:

Sex: female
Duration of test: 20th day of gestation

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

no

All female rats survived to sacrifice and no maternal toxicity was  observed. The were no differences between treated and control animals in  any of the rerpoductive endpoints investigated (corpora lutea, pre & post  implantation sites, early & late resorption sites).  The litter size,  foetal weight and sex ratio observed in treated groups was comparable to  the control group.  There were no unusual macroscopic findings among  foetuses. Microscopic examination did not show any increased incidence of  anomalies in skeletal or soft tissues. See above chapter 5.8.1 for  further details.

Conclusions:

1000 mg/kg/day is the NOAEL for maternal toxicity, teratogenicity and foetotoxicity in rats receiving behenyl alcohol by gavage for 15 days premating, during mating and up until gestation day 17. This is based on the absence of adverse effects in any of the parental, reproductive or foetal parameters examined.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-01-07 to 1994-11-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

: Analytical verification (homogeneity, stability and concentration) of the test material formulations was not performed.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD® BR rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation (females): 13 weeks
- Weight at study initiation (females): minimum 220 g (215 to 291 g on day 0 of gestation)
- Fasting period before study: no
- Housing: wire-mesh cage
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 67°F to 74°F
- Humidity (%): 28% to 58%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 1994-01-11 to 1994-02-4

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized water, prepared on site

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of test material, alkyl potassium phosphate, was weighed for each group into a tared precalibrated storage container. A sufficient amount of vehicle, deionized water, was added to bring the suspension to the appropriate volume. The preparations were stirred using a magnetic stir bar continuously during the dosing procedure. All dosing preparations were visually inspected by the Study Director on the first day of administration and appeared to be homogeneous and acceptable for administration. Preparations for all dose groups were made daily and were stored at room temperature. Analytical chemistry determinations to establish homogeneity, stability and concentration were not required by the protocol and were not conducted.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Dosage calculations were not adjusted for puritiy. The bulk material was 36.1% pure (as the potassium salt).

Details on mating procedure:

- Impregnation procedure: cohabitation
- If cohoused:
- M/F ratio per cage: 1 to 1
- Length of cohabitation: not given - Each mating pair was examined daily. Positive evidence of mating was confirmed by the presence of a copulatory plug or the presence of sperm in a vaginal smear. Each mating pair was examined daily. The day on which evidence of mating was identified was termed day 0 of gestation and the animals were separated.
- Verification of same strain and source of both sexes: yes
- Resident males were from the same strain and source.
- Resident males were untreated, sexually mature rats utilized exclusively for breeding
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: After mating, female rats were removed and allocated to the test groups.

Duration of treatment / exposure:

10 consecutive days initiating on gestation day 6 and continuing up to and including day 15 of gestation

Frequency of treatment:

once daily

Duration of test:

Females were sacrificed on day 21 post coitum.

No. of animals per sex per dose:

25/group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were based on the results of the dose range-finding sudy
- Rationale for animal assignment (if not random): The first mated female and the appropriate gestation day 0 designation were entered on the form and the female was assigned to group 1, the second mated female was assigned to group 2, and the third to group 3, etc. This process was continued daily until 25 females were placed into each group.
- Other: International guidelines recognize the efficacy of oral administration.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily for moribundity and mortality.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded individually from days 0 through 20 of gestation (prior to compound administration during the dosing period). On the first day of test material administration for this study, animals were observed for signs of toxicity approximately one, two and four hours following dosing. Few post-dosing clinical signs were observed two and four hours following dosing; therefore, only a one-hour post-dosing observation was scheduled for the remainder of the test material administration period.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights for all females were recorded on gestation days 0, 6-16 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (Individual food consumption was recorded on gestation days 0, 6-16 and 20.)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (Food intake was reported as g/animal/day and g/kg/day for each corresponding body weight change interval.)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No (Compound was administered via gavage)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20: animals were euthanized by carbon dioxide inhalation. Fetuses were removed by Caesarean section.
- Organs examined: Emphasis on the uterus and its contents, number and position of the fetuses in the uterus, early and late resorptions and the total number of implantation sites.
- Specimens of abnormal tissue were fixed in 10% neutral buffered formalin.

OTHER: Uteri with no macroscopic evidence of nidation were excised, opened and subsequently placed in 10% ammonium sulfide for detection of early implantation loss.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

- External examinations: Yes: all per litter;
sex, detailed external examination of each fetus included eyes, palate and external orifices and each fmding was recorded.
crown-rump measurements and any external malformations were recorded for late resorptions
- Soft tissue examinations: Yes: all per litter
- each fetus was examined viscerally by the Wilson sectioning technique; sections containing malformations were preserved in 10% neutral buffered formalin, all remaining serial sections were discarded
-examination of kidneys, grading for renal papillae development

- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter; The heads from all of the fetuses were examined by a mid-coronal slice.

Statistics:

All analyses were conducted using two-tailed tests for a minimum significance level of 5% comparing each treated group to the vehicle control group.
Chi-square test with Yates correction factor - fetal sex ratios
Fisher's Exact test - malformations and variations
Mann-Whitney U-test - early and late resorptions, dead fetuses, post-implantation losses, mean litter proportions of malformations and variations
One-way ANOVA with Dunnett's test - corpora lutea, total implantations, viable fetuses, fetal body weights, maternal body weights, weight changes, gravid
uterine weights and maternal food consumption
Kruskal-Wallis test - litter proportions of intrauterine data (considering the litter, rather than the fetus, as the experimental unit)

Indices:

fetal sex ratios
malformations and variations
early and late resorptions
dead fetuses
post-implantation losses
mean litter proportions of malformations and variations
corpora lutea
total implantations
viable fetuses
fetal body weights
maternal body weights
weight changes
gravid uterine weights
maternal food consumption
litter proportions of intrauterine data (considering the litter, rather than the fetus, as the experimental unit)

Historical control data:

yes (for embryotoxic effects)

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: no treatment related effects

Details on maternal toxic effects:
CUNlCAL OBSERVATIONS AND SURYIVAL No mortality. The predominant clinical sign in the treated groups was rales, which generally did not persist past one hour following dosing. In the 180.5 and 361 mg/kg/day groups, rales were observed in 17 and 16 animals, respectively, on one to five occasions one hour following dosing. Other clinical signs in the treated groups occurred infrequently (generally in single animals) and/or were observed similarly in the control group. BODY WEIGHTS AND GRAYID UTERINE WEIGHTS Mean gravid uterine weight, net body weight and net body weight gain in the highest dose group were slightly lower than the control group values. None of the differences were statistieally significant. Mean body weights, body weight gains, gravid uterine weights, net body weights and net body weight gains in the 36.1 and 180.5 mg/kg/day groups were comparable to the control group values. FOOD CONSUMPTION Food consumption, evaluated as g/animal/day and g/kg/day, in the 36.1, 180.5 and 361 mg (a.i.)/kg/day groups was unaffected by compound administration. The only statistically significant difference (p <0.01) between the control and treated groups was a slightly lower g/animall/day food consumption value in the 361 mg/kg/day group during the post-treatment period (gestation days 16-20). The difference between the control and high dose groups was slight (2 g/animal/day) and no relationship to treatment was apparent. NECROPSY DATA At the scheduled necropsy, no internal findings related to compound administration were observed at any dose level. GESTATION DAY 20 LAPAROHYSTERECTOMY DATA Intrauterine growth and survival were unaffected by compound administration at any dose level. Postimplantation losses in the treated groups were comparable to the control group value. The mean numbers of viable fetuses in the treated groups were also comparable to the control group value with one exception. The mean number of viable fetuses in the 361 mg/kg/day group (12.3 fetuses/dam) was slightly lower than the control group value (13.9 fetuses/dam). The low number of viable fetuses in the high dose group was due to lower mean numbers of corpora lutea and implantation sites in the 361 mg/kg/day group (15.9 corpora lutea/dam and 13.1 implantation sites/dam) than in the control group (17.5 corpora lutea/dam and 14.7 implantation sites/dam). As the numbers of corpora lutea and implantation sites are inherently determined prior to the initiation of dosing, no relationship to treatment was evident. The mean numbers of corpora lutea and implantation sites in the 36.1 and 180.5 mg/kg/day groups were comparable to the control group values. Mean fetal body weights and fetal sex ratios in all treated groups were comparable to the control group values. None of the differences between the control and treated groups were statistically significant.

Dose descriptor:

NOEL

Effect level:

36.1 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

361 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

361 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: no treatment related effects

Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for fetal morphological examination were 305(22), 266(19), 330(23) and 271(22) in the control, 36.1, 180.5 and 361 mg/kg/day groups, respectively. The number of fetuses (litters) with malformations were 1(1), 1(1), 1(1), 0(0) in these dose groups, respectively. EXTERNAL MALFORMATIONS AND VARIATIONS one fetus in the 180.5 mg/kg/day group had multiple anomalies. These consisted of craniorachischisis, facial cleft, microphthalmia (unilateral) and ablepharia (bilateral). No other external malformations and no external developmental variations were observed at any dose level. VISCERAL MALFORMATIONS AND VARIATIONS One fetus in the control group had an encephalomeningocele (portion of brain and meninges protruded through an opening in the skull). One fetus in the 36.1 mg/kg bw/day group was hydrocephalic. No other soft tissue malformations were observed at any dose level. Soft tissue developmental variations were observed in two control group fetuses. Both fetuses had renal papillae that were not developed and one fetus also had a distended ureter. No other soft tissue developmental variations were noted. SKELETAL MALFORMATIONS AND VARIATIONS One fetus in 180.5 mg/kg/day group had multiple external skeletal malformations and (external: craniorachischisis, facial cleft, unilateral microphthalmia and bilateral ablepharia; skeletal malformations: malformed clavicle and scapula and a vertebral anomaly with an associated rib anomaly). Skeletal developmental variations in all treated groups, such as cervical centrum no. 1 ossified, sternebrae nos. 5 and/or 6 unossified and 14th rudimentary ribs, occurred at a similar frequency in the control group, did not occur in a dose related manner and/or were within the range of historical control data. All other skeletal variants occurred infrequently (generally in single fetuses) or occurred similarly in the control group.

Dose descriptor:

NOEL

Effect level:

361 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOEL

Effect level:

361 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: fetotoxicity

Dose descriptor:

NOEL

Effect level:

361 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

CLINlCAL OBSERVATIONS AND SURYIVAL

All animals survived to the scheduled necropsy on gestation day 20. The predominant clinical sign in the treated groups was rales. In the 180.5 and 361 mg/kg/day groups, rales were observed in 17 and 16 animals, respectively, on one to five occasions one hour following dosing between gestation days 6 and 15.

Based on findings two and four hours following dosing on the initial day of compound administration, rales generally did not persist past one hour following dosing. Several animals in the 180.5 and 361 mg/kg/day groups (6 and 2, respectively) were also noted to have rales at the daily examinations prior to dosing. Rales were also observed on one occasion in a single 36.1 mg/kg/day group animal (one hour following dosing on gestation day 8). However, the single occurrence of rales at a dose level of 36.1 mg/kg/day was not suggestive of a relationship to compound administration.

Other clinical signs in the treated groups, such as hair loss on various body surfaces, red material around the nose and soft stool, occurred infrequently (generally in single animals) and/or were observed similarly in the control group. No relationship to compound administration was evident.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS

Mean body weight gains in the 361 mg/kg/day group were comparable to the control group values during the first six days of dosing (gestation days 6-9 and 9-12).

Throughout the remainder of the treatment period (gestation days 12-16), mean body weight gain in this group was slightly reduced when compared to the control group value. The difference was not statistically significant. When the overall treatment period was evaluated (gestation days 6-16), a statistically significant (P<0.05) reduced mean body weight gain occurred in the 361 mg/kg/day group when compared to the control group value. During the post-treatment period (gestation days 16-20), mean body weight gain in the 361 mg/kg/day group continued to be slightly lower than the control group value. The difference was not statistically significant. Mean body weights in the 361 mg/kg/day group were similar to the control group values throughout the study. Mean gravid uterine weight, net body weight and net body weight gain in this group were slightly lower than the control group values. None of the differences were statistically significant. Mean body weights, body weight gains, gravid uterine weights, net body weights and net body weight gains in the 36.1 and 180.5 mg/kg/day groups were comparable to the control group values.

FOOD CONSUMPTION

Food consumption, evaluated as g/animal/day and g/kg/day, in the 36.1, 180.5 and 361 mg/kg/day groups was unaffected by compound administration. The only statistically significant difference (p <0.01) between the control and treated groups was a slightly lower g/animal/day food consumption value in the 361 mg/kg/day group during the post-treatment period (gestation days 16-20). The difference between the control and high dose groups was slight (2 g/animal/day) and no relationship to treatment was apparent.

NECROPSY DATA

At the scheduled necropsy, no internal findings related to compound administration were observed at any dose level. One female in each of the control and 180.5 mg/kg/day groups (nos. 20671 and 20570, respectively) had dilated renal pelves. The 180.5 mg/kg/day group female (no. 20566) also had enlarged placentas at site nos. 7 and 12. Female no. 20656 in the 36.1 mg/kg/day group had reddened cortieo-medullary junctions in the kidneys. Control group female no. 20593 had a thickened urinary bladder containing one white calculus. All other animals were internally normal.

GESTATION DAY 20 LAPAROHYSTERECTOMY DATA

Intrauterine growth and survival were unaffected by compound administration at any dose level. Postimplantation losses in the treated groups were comparable to the control group value. The mean numbers of viable fetuses in the treated groups were also comparable to the control group value with one exception. The mean number of viable fetuses in the 361 mg/kg/day group (12.3 fetuses/dam) was slightly lower than the control group value (13.9 fetuses/dam). The low number of viable fetuses in the high dose group was due to lower mean numbers of corpora lutea and implantation sites in the 361 mg/kg/day group (15.9 corpora lutea/dam and 13.1 implantation sites/dam) than in the control group (17.5 corpora lutea/dam and 14.7 implantation sites/dam). As the numbers of corpora lutea and implantation sites are inherently determined prior to the initiation of dosing, no relationship to treatment was evident. The mean numbers of corpora lutea and implantation sites in the 36.1 and 180.5 mg/kg/day groups were comparable to the control group values. Mean fetal body weights and fetal sex ratios in all treated groups were comparable to the control group values. None of the differences between the control and treated groups were statistically significant.

FETAL MORPHOLOGICAL DATA

The numbers of fetuses (litters) available for fetal morphological examination were 305(22), 266(19), 330(23) and 271(22) in the control, 36.1, 180.5 and 361 mg/kg/day groups, respectively. The number of fetuses (litters) with malformations were 1(1), 1(1), 1(1), 0(0) in these same dose groups, respectively.

EXTERNAL MALFORMATIONS AND VARIATIONS

At the external examination, one fetus in the 180.5 mg/kg/day group (no. 20652-19) had multiple anomalies. These consisted of craniorachischisis, facial cleft, microphthalmia (unilateral) and ablepharia (bilateral). No other external malformations and no external developmental variations were observed at any dose level.

VISCERAL MALFORMATIONS AND VARIATIONS

Upon soft tissue examination, fetus no. 20661-7 in the 36.1 mg/kg/day group was hydrocephalic. The anomaly consisted of increased cavitation of the lateral and third ventricles. Control group fetus no. 20543-6 was observed with an encephalomeningocele (portion of brain and meninges protruded through an opening in the skull). No other soft tissue malformations were observed at any dose level.

Soft tissue developmental variations were observed in two control group fetuses (nos. 20655-10 and 20662-4). Both fetuses had renal papillae that were not developed and fetus no. 20662-4 also had a distended ureter. No other soft tissue developmental variations were noted.

SKELETAL MALFORMATIONS AND VARIATIONS

Multiple skeletal malformations were observed in a single fetus (no. 20652-19) in the 180.5 mg/kg/day group. This was the same fetus in which multiple malformations were noted at the external examination. The skeletal malformations consisted of a malformed clavicle and scapula and a vertebral anomaly with an associated rib anomaly (vertebral arches and/or ribs fused, extra, thickened or absent). No other skeletal malformations were observed at any dose level.

Skeletal developmental variations in all treated groups, such as cervical centrum no. I ossified, sternebrae nos. 5 and/or 6 unossified and 14th rudimentary ribs, occurred at a similar frequency in the control group, did not occur in a dose related manner and/or were within the range of the WIL historical control data. All other skeletal variants occurred infrequently (generally in single fetuses) or occurred similarly in the control group.

SUMMARY OFEXTERNAL. VISCERAL AND SKELETAL EXAMINATIONS

Fetal external, soft tissue, and skeletal malformations were observed in 1(1), 1(1), 1(1) and 0(0) fetuses (litters) in the control, 36.1, 180.5 and 361 mg/kg/day groups, respectively, and were considered to be spontaneous in ongm. No treatment related trends were apparent when either the actual numbers of malformations were compared or when the specific types of anomalies expressed were considered. None of the values in the treated groups were statistically significant when compared to the control group values. Fetal developmental variations in the treated groups were observed at a frequency similar to that in the control group, occurred infrequently or were within the range of the WILL historical control data.

Conclusions:

In this study administration of Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts to 25 female Sprague-Dawley Crl:CD® BR rats/dose by oral gavage from day 6 through 15 of gestation had no compound related effects up to and including the highest tested dose of 361 mg/kg/day (active ingredient). Intrauterine growth and survival were unaffected by treatment at any dose level. The fetal malformations observed in this study were all considered to be spontaneous in origin. No treatment-related fetal developmental variations were observed at any dose level.

Executive summary:

In a developmental toxicity study according to OECD guideline 414, Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts (36.1% a.i.) was administered to 25 female Sprague-Dawley Crl:CD® BR rats/dose by oral gavage at dose levels of 0 (control), 36.1, 180.5 and 361 mg/kg bw/day (active ingredient) from day 6 through 15 of gestation. Females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section.

No treatment-related effects on mortality, number of abortions, clinical signs, food consumption, gross pathology, intrauterine growth and survival (incl. postimplantation loss, viable litter size, mean fetal body weights, fetal sex ratios, mean numbers of corpora lutea and implantation sites) were observed.

No treatment-related fetal malformations or fetal developmental variations were observed at any dose level.

The NOEL for maternal toxicity is 36.1 mg/kg bw/day.

The NO(A)EL for maternal toxicity is 361 mg/kg bw/day (highest dose administered).

The NOEL for developmental toxicity is 361 mg/kg bw/day (highest dose administered).

The NOEL for embryotoxicity is 361 mg/kg bw/day (highest dose administered).

The NOEL for fetotoxicity is 361 mg/kg bw/day (highest dose administered).

The NOEL for teratogenicity is 361 mg/kg bw/day (highest dose administered).

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rat.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

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

No data are available with C20-22 alkyl phosphate regarding the developmental toxicity / teratogenicity endpoint. Thus it was evaluated based on data available on related substances.

The developmental toxicity of C20-22 alkyl phosphate was evaluated based on 3 studies:

-       A prenatal developmental toxicity study with Alkyl Potassium Phosphate (C9-11 branched and linear alkyl esters) performed in rats by oral route according to OECD 414 and in compliance with GLP. This study was scored as validity 2 according to Klimisch criteria.

-       A prenatal developmental toxicity study with docosan-1-ol performed in rats by oral route, similar to guideline. This study was scored as validity 2 according to Klimisch criteria.

-       A prenatal developmental toxicity study with docosan-1-ol performed in rabbits by oral route according to ICH guideline. This study was scored as validity 2 according to Klimisch criteria.

Study with Alkyl Potassium Phosphate (rat, oral):

In a developmental toxicity study according to OECD guideline 414, Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts (36.1% a.i.) was administered to 25 female Sprague-Dawley Crl:CD® BR rats/dose by oral gavage at dose levels of 0 (control), 36.1, 180.5 and 361 mg/kg bw/day (active ingredient) from day 6 through 15 of gestation. Females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section.

No treatment-related effects on mortality, number of abortions, clinical signs, food consumption, gross pathology, intrauterine growth and survival (incl. post-implantation loss, viable litter size, mean fetal body weights, fetal sex ratios, mean numbers of corpora lutea and implantation sites) were observed.

No treatment-related fetal malformations or fetal developmental variations were observed at any dose level.

The NO(A)EL for maternal toxicity is 361 mg/kg bw/day (highest dose administered).

The NOEL for developmental toxicity, embryotoxicity, fetotoxicity and teratogenicity is 361 mg/kg bw/day (highest dose administered).

Study with docosan-1-ol (rat, oral):

In this study, docosan-1-ol was administered to female Sprague-Dawley Crl:CD® rats by oral gavage at dose levels of 0 (control), 10, 100 and 1000 mg/kg bw/day for 15 days prior to mating, during mating and up to Day 17 of gestation.

1000 mg/kg/day is the NOAEL for maternal toxicity, teratogenicity and foetotoxicity in rats receiving behenyl alcohol by gavage for 15 days premating, during mating and up until gestation day 17. This is based on the absence of adverse effects in any of the parental, reproductive or foetal parameters examined.

Study with docosan-1-ol (rabbit, oral):

In this ICH study (similar to OECD 414 guideline), docosan-1-ol was administered to female New-Zealand rabbits by oral gavage at dose levels of 0 (control), 125, 500 and 2000 mg/kg bw/day from day 6 through 19 of gestation.

The NOAEL for maternal toxicity, teratogenicity and foetotoxicity in rabbits was 2000 mg/kg/day (highest dose tested).

Justification for classification or non-classification

Based on the classification criteria of UN/EU GHS, and given the absence of signs of toxicity, no classification for reproductive and developmental toxicity is warranted.