Phosphoric acid, mono- and di-C16-18-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study, tested with the source substance Phosphoric acid, dodecyl ester, sodium salt. 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.

Reason / purpose for cross-reference:

reference to same study

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

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

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

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

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

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

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)
- 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

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

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is of high quality with Klimisch score = 1; the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reliable, relevant and adequate data on reproductive toxicity are available for the read-across substance Phosphoric acid, dodecyl ester, sodium salt.

The read-across approach is appropriate due to similar composition of source substance and registered substance. From the available data is can be concluded that the repeated dose toxicity of substances with different alkyl moieties (C12, C14, C9-15 linear and branched) is comparable.

Phosphoric acid alkyl esters are hydrolysed unspecifically by phosphatases, e.g. acid phosphatase or alkaline phosphatase. Both enzymes are found in most organisms from bacteria to human. Alkaline phosphatases are present in all tissues, but are particularly concentrated in liver, kidney, bile duct, bone, placenta. In human and most other mammals three isoenzymes of Alkaline phosphatase exist: intestinal ALP, placental ALP, tissue non-specific ALP (present in bone, liver, kidney, skin).

Seven different forms of Acid phosphatase are known in humans and other mammals. These are also present in different tissues and organs (predominantly erythrocytes, liver, placenta, prostate, lung, pancreas).

Linear and branched primary aliphatic alcohols are oxidised to the corresponding carboxylic acid, with the corresponding aldehyde as a transient intermediate. The carboxylic acids are further degraded via acyl-CoA intermediates in by the mitochondrial beta-oxidation process. Branched aliphatic chains can be degraded via alpha- or omega-oxidation (see common text book on biochemistry).

“The long chain aliphatic carboxylic acids are efficiently eliminated and aliphatic alcohols are therefore not expected to have a tissue retention or bioaccumulation potential (Bevan, 2001).

Longer chained aliphatic alcohols within this category may enter common lipid biosynthesis pathways and will be indistinguishable from the lipids derived from other sources (including dietary glycerides) (Kabir, 1993; 1995a,b).

A comparison of the linear and branched aliphatic alcohols shows a high degree of similarity in biotransformation. For both sub-categories the first step of the biotransformation consists of an oxidation of the alcohol to the corresponding carboxylic acids, followed by a stepwise elimination of C2 units in the mitochondrial β-oxidation process. The metabolic breakdown for both the linear and mono-branched alcohols is highly efficient and involves processes for both sub-groups of alcohols. The presence of a side chain does not terminate the β-oxidation process, however in some cases a single Carbon unit is removed before the C2 elimination can proceed.” (OECD SIDS, 2006).

 

Thus, it can be expected, that also the effects on reproduction of the Phosphoric acid C12-alkyl ester will be similar to those of Phosphoric acid, mono- and di- C16-18 (even numbered) alkyl esters.

 

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/d.

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 behaviour 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 behaviour 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 bw/d had no effect on the development.

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

Higher-tier fertility study (two-generation study) is not required at this tonnage band, since there were no adverse effects observed in the repeated dose toxicity study in reproductive organs or tissues or any adverse effects in the screening studies for reproductive toxicity (OECD 422). Therefore, there is no data gap in fertility. There is no reason to believe that results of the screening study would not be relevant for fertility in humans and therefore, for risk assessment.

Short description of key information:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test oral (gavage), rat (Sprague-Dawley) m/f (OECD guideline 422, GLP no data): reproduction, breeding and developmental NOEL: 1000 mg/kg bw/day (both sexes); read-across substance Phosphoric acid, dodecyl ester, sodium salt

Justification for selection of Effect on fertility via oral route:
OECD guideline study, no deviations, GLP

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity study: rat (Sprague-Dawley Crl:CD® BR),  gavage, (OECD 414): NOEL developmental toxicity, embryotoxicity, fetotoxicity and teratogenicity 361 mg/kg bw/d (highest dose administered).  No compound related developmental toxicity, embryotoxicity, fetotoxicity or teratogenicity has been observed in a developmental toxicity study with Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts.  

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

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.

Remarks:

Doses / Concentrations:
0 mg/kg bw/day (vehicle control)
Basis:
actual ingested
active ingredient

Remarks:

Doses / Concentrations:
36.1 mg/kg bw/day
Basis:
actual ingested
active ingredient

Remarks:

Doses / Concentrations:
180.5 mg/kg bw/day
Basis:
actual ingested
active ingredient

Remarks:

Doses / Concentrations:
361 mg/kg bw/day
Basis:
actual ingested
active ingredient

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

Dose descriptor:

NOAEL

316 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is of high quality with Klimisch score = 1; the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

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

Reliable, relevant and adequate data on prenatal developmental toxicity are available for the read-across substance Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts.

The read-across approach is appropriate due to similar composition of source substance and registered substance. From the available data is can be concluded that the repeated dose toxicity of substances with different alkyl moieties (C12, C14, C9-15 linear and branched) is comparable.

Phosphoric acid alkyl esters are hydrolysed unspecifically by phosphatases, e.g. acid phosphatase or alkaline phosphatase. Both enzymes are found in most organisms from bacteria to human. Alkaline phosphatases are present in all tissues, but are particularly concentrated in liver, kidney, bile duct, bone, placenta. In human and most other mammals three isoenzymes of Alkaline phosphatase exist: intestinal ALP, placental ALP, tissue non-specific ALP (present in bone, liver, kidney, skin).

Seven different forms of Acid phosphatase are known in humans and other mammals. These are also present in different tissues and organs (predominantly erythrocytes, liver, placenta, prostate, lung, pancreas).

Linear and branched primary aliphatic alcohols are oxidised to the corresponding carboxylic acid, with the corresponding aldehyde as a transient intermediate. The carboxylic acids are further degraded via acyl-CoA intermediates in by the mitochondrial beta-oxidation process. Branched aliphatic chains can be degraded via alpha- or omega-oxidation (see common text book on biochemistry).

“The long chain aliphatic carboxylic acids are efficiently eliminated and aliphatic alcohols are therefore not expected to have a tissue retention or bioaccumulation potential (Bevan, 2001).

Longer chained aliphatic alcohols within this category may enter common lipid biosynthesis pathways and will be indistinguishable from the lipids derived from other sources (including dietary glycerides) (Kabir, 1993; 1995a,b).

A comparison of the linear and branched aliphatic alcohols shows a high degree of similarity in biotransformation. For both sub-categories the first step of the biotransformation consists of an oxidation of the alcohol to the corresponding carboxylic acids, followed by a stepwise elimination of C2 units in the mitochondrial β-oxidation process. The metabolic breakdown for both the linear and mono-branched alcohols is highly efficient and involves processes for both sub-groups of alcohols. The presence of a side chain does not terminate the β-oxidation process, however in some cases a single Carbon unit is removed before the C2 elimination can proceed.” (OECD SIDS, 2006).

The Phosphoric acid alkyl esters with branched fatty alcohol chains can be considered as a worst case scenario because the metabolism of the resulting branched fatty acids occurs less efficient compared to linear fatty acids.

 

Thus, it can be expected, that also the effects on development of the Phosphoric acid C9-15, branched and linear alkyl ester will be similar to those of Phosphoric acid, mono- and di- C16-18 (even numbered) alkyl esters.

 

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/d (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/d.

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

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

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

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

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

There is no data gap in prenatal developmental toxicity. There is no reason to believe that results of the prenatal developmental toxicity study would not be relevant for developmental toxicity in humans and, therefore, for risk assessment.

 

Justification for selection of Effect on developmental toxicity: via oral route:
OECD guideline study, GLP

Justification for classification or non-classification

In conclusion, the results of the available data on toxicity to reproduction, developmental toxicity and teratogenicity indicate that Phosphoric acid, mono- and di- C16-18 (even numbered) alkyl esters do not need to be classified for toxicity to reproduction (fertility), developmental toxicity and teratogenicity according to Directive 67/548/EEC as well as CLP, EU GHS (Regulation 1272/2008/EC) and therefore labelling is not necessary.