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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphoric acid, dodecyl ester, sodium salt
EC Number:
256-865-1
EC Name:
Phosphoric acid, dodecyl ester, sodium salt
Cas Number:
50957-96-5
IUPAC Name:
sodium dodecyl hydrogen phosphate
Constituent 2
Reference substance name:
50957-96-5 (Phosphoric acid, dodecyl ester, sodium salt)
IUPAC Name:
50957-96-5 (Phosphoric acid, dodecyl ester, sodium salt)
Details on test material:
- Name of test material (as cited in study report): Phosphoric acid, dodecyl ester, sodium salt
- Physical state: solid
- Analytical purity: not given in test report, but according to supplier 100%

Test animals

Species:
rat
Strain:
other: Sprague-Dawley SPF (Crj;CD (SD) IGS
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: 338 to 395 g (males), 219 to 256 g (females)
- Housing:
- individually in bracket type metal wire cages (250 mm [W] × 350 mm [D] × 200 mm [H];
- during mating: one pair of male and female animals per cage
- from gestation day 17 to day 5 of lactation: individual housing in a plastic Econ cage (340 mm [W] × 400 mm [D] × 185 mm [H]) with bedding
- Diet (e.g. ad libitum): Solid chow NMF, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C to 26°C
- Humidity (%):37% to 77%
- Air changes (per hr): 10 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
the test substance was suspended in olive oil in an agate mortar;
prepared at a frequency of at least once every 7 days and stored in refrigerator (observed temperature: 3°C-5°C) in brown glass bottles until use

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil; no justification given on choice
- Concentration in vehicle: 50, 100, and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight, exact amount based on latest body weight
- Lot/batch no. (if required): Maruishi Pharmaceutical Co., Ltd., batch No. 4604, 4720
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1 male + 1 female from same dosing group
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how):
- individually in a bracket type metal wire cage
- from gestation day 17 to day 5 of lactation, the animals were housed individually in a plastic Econ cage with bedding
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- suspensions of each concentration used for administration at week 1 and on the last week of
administration were analyzed by HPLC
- for all suspensions tested, the percentage of the test substance relative to the nominal value was in the range of 96.5% to 105.0%, with a C.V. in the range of 1.0% to 5.3%, which were within the
acceptable range
Duration of treatment / exposure:
Males: 42 days total; 14 days before mating, through the mating period, up to 1 day before necropsy
Females: 42 to 45 days total; 14 days before mating, through the mating and gestation period, up to day 4 of lactation
Offspring: no treatment
Frequency of treatment:
Once daily for 7 days per week
Details on study schedule:
- Parturition: The females were allowed to Iitter normally. Offspring was kept with the dam until termination
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 males, 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a preliminary 14-day repeated-dose oral toxicity study (doses: 125, 250, 500, 1000 mg/kg, administration of the test substance did not produce any effect up to 1000 mg/kg. Therefore, 1000 mg/kg was set as the highest dose, and doses of 500 and 250 mg/kg were derived by dividing by a common factor of 2.

- Rationale for animal assignment (if not random):
Animals were stratified according to the body weight on the day of group assignment (1 day before
the start of administration), and assigned to each group in such a way that the mean body weight was
as close as possible among the groups. The assignment of individual animals was performed by a
combination of the block placement method and random sampling.

- Section schedule rationale (if not random): not given
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
external appearance, nutritional condition, posture, behavior, and excrements, 3 times daily
(before, immediately after, and 2 hours after the administration) during the administration period and
once every morning during the recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
before the start of administration
males: once every week during administration
females: once every week during pre-mating and mating period, days 1, 7, 14 and 20 of gestation, day 4 of lactation
recovery groups: once every week during administration and recovery

BODY WEIGHT: Yes
- Time schedule for examinations:
males, main group: days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration, day of necropsy
females, main group: days 1, 4, 8, 11 and 15 of administration, days 0, 4, 7, 11, 14, 17 and 20 of gestation, days 0 and 4 of lactation
males + females, recovery group: additional days 1, 4, 8, 11 and 14 of the recovery period, day of necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

males, main group: days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration
females, main group: days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20 of gestation, days 2 and 4 of lactation
males+females, recovery group: additional on days 1, 4, 8, 11 and 14 of the recovery period

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: Yes
- Time schedule for examinations: week 6 of administration

OTHER:
HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS, NEUROBEHAVIOURAL EXAMINATION
see detailed study description under chapter 7.5.1 "Repeated dose toxicity: oral"
Oestrous cyclicity (parental animals):
During pre-mating, vaginal smear profile was classified into proestrus, estrus, metestrus, and anestrus, from which frequency of the estrus and the days from one estrus to the next estrus (estrous cycle) were calculated.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, body weight (day 0 and 4 of lactation), gross necropsy on day 4 of lactation

GROSS EXAMINATION OF DEAD PUPS:
no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, day 43 (one day after last administration)
- Maternal animals: All surviving animals, day 46 (one day after last administration)

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
- The F1 offspring were sacrificed at day 4 of lactation
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed
Statistics:
Bartlett test for homoscedasticity (2-sided significance level, 0.01), Dunnett’s test if homoscedastic, otherwise Dunnett-type test (2-sided significance level 0.05 and 0.01):
body weight, food consumption, water intake, number of occurrences of the estrus profile, estrous cycle, days to copulation, gestation period, number of corpora lutea, number of implantation sites, number of live-born pups, open field observations (defecating frequency, standing frequency), function tests (landing foot splay), grip strength and spontaneous motor activity, quantitative urinalysis parameters, hematological tests, blood chemistry tests, organ weight;
mean body weight of the pups, implantation rate, stillbirth rate, live birth rate, external anomaly rate, and viability rate of the pups for each mother animal,

χ2 test with Yates’ continuity correction (2-sided significance level, 0.05 and 0.01), or if there were cells with an expected frequency of 5 or less, Fisher’s exact test (2-sided significance level, 0.05 and 0.01):
copulation rate, fertility rate, conception rate, delivery rate, sex ratio of the pups, auditory response, approach reaction, contact reaction, pain reaction, pupillary reflex, and air righting reflex, the number of animals that copulated, the number of pregnant animals, the number of females that gave birth to live pups, the number of live male pups, the number of live female pups, and the number of animals that showed normal reflexes
Reproductive indices:
Copulation index: (number of copulated animals/number of mated animals) x 100
Insemination index: (number of males which inseminated females / number of copulated males) x 100
Fertility index: (number of pregnant females / number of copulated females) x 100
Offspring viability indices:
Viability index on day 4 of lactation: (number of live pups on day 4 / number of live pups on day 0) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- no mortality occurred during study period
- no abnormalities in home cage observations
- one female (500 mg/kg) showed opacity of an eyeball (unilateral) from gestation day 5, which was not related to the dose and was therefore considered to be an incidental change
- males of the 1000 mg/kg group showed a significant increase of the defecation frequency during weeks 1 and 2 of administration, which was a very mild transient change and considered to be within normal range


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- a significantly greater increase in body weight in the females of the 250 and 1000 mg/kg groups during the lactation period, but not dose-related
- a significant increase was observed on days 2 and 4 of lactation in the females of the 250 mg/kg dose group, but not dose-related

- one male (1000 mg/kg, recovery group; details in chapter 7.5.1 "Repeated dose toxicity: oral") showed decreased body weight gain during the administration period and decreased body weight during the recovery period; decreased spontaneous activity; wheezing in the observation of the general condition.
- the body weights of the other 4 males and 5 females in the same group were similar to those of the animals in the control group, showing no statistically significant differences


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant difference in any of the treated groups from control group


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
not examined


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- one pair in the 500 mg/kg group did not copulate
- copulation occurred in all of the other pairs by day 4 after the start of mating, resulting in pregnancy in all of the females that copulated.
- significant increases in the number of corpora lutea and live-born pups were observed in the 250 mg/kg group, but not dose-related

- no abnormalities observed in nest building, pup gathering, or lactating behavior in any of the mother animals

- no significant differences in the days to copulation, copulation rate, fertility rate, conception rate, delivery rate, gestation period, number of corpora lutea, number of implantation sites, implantation rate, stillbirth rate, number of live-born pups, and live birth rate between the control group and any of the treatment groups


ORGAN WEIGHTS (PARENTAL ANIMALS)
- at 500 mg/kg males showed a significant decrease in absolute and relative thymus gland weight
- at 500 mg/kg males showed a significant increase in relative testis weight
- at 500 mg/kg females showed a significant increase in absolute heart weight
- those changes were not dose-related and are therefore considered to be of no toxicological relevance
- at 1000 mg/kg females of the recovery group showed a significant decrease in absolute and relative thyroid gland weight; no such change was observed at the end of the administration period


GROSS PATHOLOGY (PARENTAL ANIMALS)
- indentation of the anterior stomach was observed in 0 (250 mg/kg), 5 (500 mg/kg) and 7 (1000 mg/kg) males, and 1 (250 mg/kg), 1 (500 mg/kg) and 3 (1000 mg/kg)females
- white foci in the anterior stomach were observed in 1 male of the 500 mg/kg group
- rough mucosa in the anterior stomach was observed in 5 (500 mg/kg) and 9 (1000 mg/kg) males, and 5 (500 mg/kg) and 6 (1000 mg/kg) females
- indentation of the glandular stomach was observed in 1 female of the 500 mg/kg group
- dark red foci in the glandular stomach were observed in 1 (250 mg/kg), 2 (500 mg/kg) and 1 (1000 mg/kg) males and 4 (control), 2 (250 mg/kg), 1 (500 mg/kg) and 1 (1000 mg/kg) females

- renal pelvis dilation was observed in 2 (250 mg/kg) and 1 (1000 mg/kg) males
- unilateral corneal opacity was observed in 1 female of the 500 mg/kg group

- rough mucosa in the anterior stomach was observed in 1 male of the 1000 mg/kg group at the end of the recovery period. This animal also showed expansion of the digestive tract from the stomach to the colon due to gas accumulation, and a mild decrease in the size of the testis.
- dark red foci were observed in the lung of 1 female of the 1000 mg/kg group at the end of the recovery period


HISTOPATHOLOGY (PARENTAL ANIMALS)
Administration of the test substance had effects on the anterior stomach of the animals in the 250 mg/kg and higher dose groups:
- mild to moderate erosions or ulcers of the anterior stomach were observed in 4 (500 mg/kg) and 4 (1000 mg/kg) males and 1 (250 mg/kg), 1 (500 mg/kg) and 1 (1000 mg/kg) females
- very mild to moderate thickening of the anterior stomach mucosa was observed in 1 (250 mg/kg), 4 (500 mg/kg) and 5 (1000 mg/kg) males and 1 (250 mg/kg), 4 (500 mg/kg) and 3 (1000 mg/kg)
females
- very mild to mild edema of the submucosal tissue in the anterior stomach was observed in 1 (250 mg/kg), 5 (500 mg/kg) and 5 (1000 mg/kg) males and 4 (500 mg/kg) and 3 (1000 mg/kg) females
Most of these changes in the anterior stomach were localized findings.

- At the end of the recovery period, moderate thickening of the anterior stomach mucosa was observed in 1 male of the 1000 mg/kg group.

All other findings observed were considered to be incidental changes as judged from the frequency of their occurrence:
- Epididymis: very mild infiltration by stromal cells was observed in 1 male of the control group.
- Heart: very mild localized myocarditis was observed in 4 males of the control group and 1 male of
the 1000 mg/kg group.
- Kidney: very mild basophilic tubules were observed in 3 males of the control group and 1 male and
1 female in the 1000 mg/kg group.
- Liver: very mild, minute granulomas were observed in 3 males of the control group and 1 male of
the 1000 mg/kg group.
- Lung (including bronchi): very mild mineral deposits in the arterial walls were observed in 1 male of
the control group and 1 female of the 1000 mg group. Very mild accumulation of foam cells was
observed in 2 males and 1 female of the control group, and 1 male and 3 females of the 1000 mg/kg
group.
- Spleen: very mild to mild extramedullary hematopoiesis was observed in 5 females each in the
control group and 1000 mg/kg group.
- Stomach: inclusion cysts were observed in 1 male of the 500 mg/kg group. Very mild to mild
erosions in the glandular stomach were observed in 1 male in the 1000 mg/kg group and 3 (control), 1 (250 mg/kg) and 1 (500 mg/kg) females

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: mating index; fertility index; number of implantation sites; duration of pregnancy; birth index; live birth index; pregnancy index; litter size; other: estrous cycle, days to copulation, copulation rate

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
- during the lactation period, death occurred in only 4 pups in the control group and 2 pups in the
1000 mg/kg group.
- no significant differences in the viability rate on day 4 of lactation between the control group and treatment groups.


CLINICAL SIGNS (OFFSPRING)
not examined


BODY WEIGHT (OFFSPRING)
- no significant differences at birth or on day 4 of lactation between the control group and any of the treatment groups


SEXUAL MATURATION (OFFSPRING)
not examined


ORGAN WEIGHTS (OFFSPRING)
not examined


GROSS PATHOLOGY (OFFSPRING)
- thymic cervical residue was observed in 1 (control), 1 (250 mg/kg), 2 (1000 mg/kg) males and 3 (control), 1 (250 mg/kg), 3 (1000 mg/kg) females
- these findings were considered not to be a sign of toxicity as no dose response relationship was noted


HISTOPATHOLOGY (OFFSPRING)
not examined


OTHER FINDINGS (OFFSPRING)
- no significant differences were observed in the sex ratio, or external anomaly rate between the control group and any of the treatment groups
- observation for external anomalies showed kinking of the tail in 1 animal of the 500 mg/kg group, which was considered to be a spontaneous occurrence as judged from the frequency of occurrence and the type of the anomaly

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: litter size; litter weight; pup weight; sex ratio; viability index; other: external anomaly rate, necropsy findings

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No reproduction, breeding and developmental toxicity was observed for treatment with Phosphoric acid, dodecyl ester, sodium salt up to 1000 mg/kg bw/day.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD guideline 422 Phosphoric acid, dodecyl ester, sodium salt was administered to 12 Sprague-Dawley rats/sex/dose by daily oral gavage at dose levels of 0 (control), 250, 500, and 1000 mg/kg bw/day.

The males were exposed 14 days before mating, through the mating period, up to 1 day before termination (42 days in total). The females were exposed 14 days before mating, through the mating and gestation period, up to day 4 of lactation (42 to 45 days in total).

Administration of the test substance did not have any effect on the estrous cycle, days to copulation, copulation rate, fertility rate, or conception rate. Similarly, administration of the test substance did not have any effect on the delivery rate, gestation period, number of corpora lutea, number of implantation sites, implantation rate, stillbirth rate, number of live-born pups, live-birth rate in the mother animals, or on the sex ratio of the littermates. No abnormalities were observed in the lactating behavior during the lactation period either. These results suggest that administration of the test substance even at 1000 mg/kg had no effect on the reproductive function, such as that shown by the copulation rate, of the males or females, or in the fertility rate, conception rate, or on the gestation maintenance, delivery, or lactating behavior in the mother animals.

Pups showed no changes caused by the administration of the test substance regarding the observation at birth, necropsy findings on day 4 of lactation, body weight, or viability rate, which suggested that administration of the test substance even at 1000 mg/kg had no effect on the development.

The reproduction, breeding and developmental NOEL is 1000 mg/kg bw/d.

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the rat is acceptable and satisfies the guideline requirements of OECD TG 422.

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