Glycerides, C12-18 mono-, di- and tri-

Toxicity to reproduction

Some information in this page has been claimed confidential.

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:

read-across source

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

Density 0.944
pH 5.7 at concentration of 1%
Vehicle Polyethylene glycol 400, specific gravity 1.125. (Merck, Darmstadt,
Germany).
Rationale for vehicle Based on trial formulations performed at NOTOX.
Method of formulation Formulations (w/w) were prepared daily within 6 hours prior to
dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944),
and the specific gravity of the vehicle (1.125).
Storage conditions At ambient temperature.

Test animals

Species:

other: Rat: Crl:Wiatar(Han) (outbred, SPF-Quality). Nulliparous and nonpregnant females and untreated animals

Strain:

other: Crl:Wistar(Han) (outbred, SPF-Quality)

Details on species / strain selection:

Nulliparous and nonpregnant
females and untreated animals

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were housed in a controlled environment, in which optimal
conditions were considered to be approximately 15 air changes per
hour, a temperature of 21 ± 3°C (actual range: 19.7 - 21.9°C), a
relative humidity of 40 - 70% (actual range: 22 - 71%) and 12 hours
artificial light and 12 hours darkness per day. Cleaning procedures
in the room might have caused the temporary fluctuations above
the optimal maximum level of 70% for relative humidity (with a
maximum of 4 hours). Temporary fluctuations from the light/dark
cycle (with a maximum of 1 hour) occurred due to performance of
pupillary reflex tests in the room. Based on laboratory historical
data, these fluctuations were considered not to have affected the
study integrity.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

Main animals and Recovery animals of both sexes were exposed
for at least 28 days including at least 2 weeks of exposure prior to
mating and during the mating period for Main males. The Repro
females were exposed for at least 14 days prior to mating, during
mating, during the post coitum and lactation periods, and up to the
day prior to necropsy.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and
females, one Repro female was cohabitated with one Main male of
the same treatment group, avoiding sibling mating (Charles River
supplied non-litter mates). Detection of mating was confirmed by
evidence of sperm in the vaginal lavage, by staging of the estrous
cycle and/or or by the appearance of an intravaginal copulatory
plug. Detection of mating was not confirmed for animal no. 98
which did deliver. The mating date of this animal was estimated at
21 days prior to the actual delivery date. This day was designated
Day 0 post-coitum. Once mating had occurred, the males and
females were separated. A maximum of 14 days was allowed for
mating. After 14 days of mating, females who had not shown
evidence of mating were separated from their males.

Details on analytical verification of doses or concentrations:

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in
agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

Duration of treatment / exposure:

10 days

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each
day with a maximum of 6 hours difference between the earliest and
latest dose. Animals were dosed up to the day prior to scheduled
necropsy.

Details on study schedule:

Experimental starting date 17 February 2010 (allocation dose range finding
study; see Appendix 5)
Start treatment 17 February 2010
Start mating 03 March 2010
Start Recovery 19 March 2010
Blood sampling of Main and Recovery
animals
Main and recovery males: 18 March 2010
Main and recovery females: 19 March 2010
Recovery: 2 April 2010
Necropsy of Main and Recovery animals Main: 18 March and 19 March 2010
Recovery: 02 April 2010
Delivery of litters 25 - 28 March 2010
Necropsy of Repro females and pups 30 March - 07 April 2010
Experimental completion date 07 April 2010 (end of in-life phase)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Control animals:

yes

Details on study design:

Study Design
Group Dose level Number of animals Animal numbers
mg/kg b.w./day F0 males F0 females males females
1 Main
1 Recovery
1 Repro
0 10
5**
--
5**
5**
10
01-10
11-15
51-55
56-60
81-90
2 Main
2 Repro
100 10 5**
10
16-25 61-65
91-100
3 Main
3 Repro
300 10 5**
10
26-35 66-70
101-110
4 Main
4 Recovery
4 Repro
1000 10
5**
--
5**
5**
10
36-45
46-50
71-75
76-80
111-120
** These animals were not mated and, consequently, were not used for the
assessment of reproduction/developmental toxicity.

Examinations

Parental animals: Observations and examinations:

Mortality / Viability At least twice daily. Animals showing pain, distress or discomfort,
which was considered not transient in nature or was likely to become
more severe, were sacrificed for humane reasons based on OECD
guidance document on humane endpoints (ENV/JM/MONO/
2000/7). The time of death was recorded as precisely as possible.
Clinical signs Detailed clinical observations were made in all animals, at least
immediately after dosing. Once prior to start of treatment and at
weekly intervals this was also performed outside the home cage in
a standard arena. Arena observations were conducted during the
treatment phase only. Arena observations were not performed
when the animals were mating, or housed individually. The time of
onset, degree and duration was recorded. All symptoms were
recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate,
grade 3 = severe, grade 4 = very severe
Functional Observations The following tests were performed on the first Main males
(randomly selected at allocation), all Recovery animals and the 5
Main females from each group:
- hearing ability, pupillary reflex, static righting reflex and grip
strength (score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 1-hour for individual
animals, using a computerized monitoring system; Pearson
Technical Services, Suffolk, Great Britain).
During the motor activity test, animals were caged individually
The assigned animals were tested during week 4 of treatment (all
before blood sampling). Since no treatment-related findings were
noted, the functional observation tests and motor activity
measurements were not extended to all animals at the end of the
recovery phase.
Body weights Males and females were weighed on the first day of exposure and
weekly thereafter. Mated Repro females were weighed on Days 0, 4,
7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and
4.
Food consumption Weekly, for males and females. Food consumption was not
recorded during the mating period (except for males). Food
consumption of mated Repro females was measured on Days 0, 4,
7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
Water consumption Subjective appraisal was maintained during the study, but no
quantitative investigation was introduced as no effect was
suspected.
General reproduction data Male number paired with, mating date, confirmation of pregnancy,
and delivery day were recorded. Pregnant females were examined
to detect signs of difficult or prolonged parturition, and cage debris of
these females was examined to detect signs of abortion or
premature birth. Any deficiencies in maternal care (such as
inadequate construction or cleaning of the nest, pups left scattered
and cold, physical abuse of pups or apparently inadequate lactation
or feeding) were examined.
For further detail on summary data, see APPENDIX 1 and on individual data, see APPENDIX 2.
Note to tables of clinical signs, body weights, food consumption and macroscopic findings: The
phases “Treatment” and “Recovery” concern the Main and Recovery animals. The phases “Pre
mating”, “Mating”, Post coitum” and “Lactation” concern the Repro females.

Statistics:

Test statistics were calculated on the basis of exact values
for means and pooled variances. Individual values, means and standard deviations may have
been rounded off before printing. Therefore, two groups may display the same printed means
for a given parameter, yet display different test statistics values

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted that were attributable to treatment with RIKEMAL PL-
012(R).
The female killed in extremis at 1000 mg/kg (no. 117) showed hunched posture, laboured
respiration, rales, piloerection, salivation and lean appearance prior to sacrifice.
Salivation seen occasionally after dosing among a few animals at 100, 300 and 1000 mg/kg/day
was considered to be a physiological response rather than a sign of systemic toxicity
considering the nature and minor severity of the effect and its time of occurrence (i.e. after
dosing).
Other incidental findings that were noted included diarrhoea, rales, red material on the snout
and alopecia, scales and/or scales. These findings occurred within the range of background
findings to be expected for rats of this age and strain which are housed and treated under the
conditions in this study. At the incidence observed, these were not considered to be signs of
toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment
with the test substance.
Female no. 117 (1000 mg/kg/day) was killed in extremis on Day 17 post-coitum. Microscopic
examination revealed a marked granuloma in the bronchus region containing macrophages
surrounding food particles and with central areas of necrosis. These findings were indicative of
a gavage accident.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were noted up to
1000 mg/kg.
Any statistically significant changes in body weight gain observed among males and females
during the treatment or recovery period were considered to be of no toxicological relevance
since the changes occurred in the absence of a dose-related trend and/or were of a slight
and/or temporary nature. These changes consisted of a higher body weight gain for males at
1000 mg/kg/day during the recovery phase, a lower body weight gain of Repro females at 1000
mg/kg/day on Day 11 of the post coitum period, and a higher body weight gain of Main females
at 100 mg/kg/day over treatment Days 8-22.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and
control animals.

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time (PT) in males at 1000 mg/kg/day and lower
relative lymphocyte counts in females at 300 mg/kg/day at the end of treatment were considered
to be of no toxicological relevance. These changes were absent at the end of the recovery
period, occurred in the absence of a (clear) treatment-related trend and remained within the
range considered normal for rats of this age and strain.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of
no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend,
remained within the range considered normal for rats of this age and strain and/or were present
at the end of the recovery period only. At the end of treatment these changes consisted of lower
alanine aminotransferase in males at 300 and 1000 mg/kg/day, and higher chloride levels in
males at 100 mg/kg/day. Changes at 1000 mg/kg/day at the end of the recovery phase included
lower albumin and higher glucose levels in females, and higher inorganic phosphate levels in
males.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. Males at 300 mg/kg had
significantly lower high sensor counts. Since the range of high sensor values encountered at
300 mg/kg/day was similar to that observed in the control group and no dose-related trend was
noted, no toxicological relevance was ascribed to this variation.
A notable variation in high sensor counts was recorded for females at 1000 mg/kg/day. Since
the range of values at this dose was essentially similar to that observed in the control group, it
was considered that no toxicologically significant effect on high sensor counts had occurred in
females at 1000 mg/kg/day

Organ weight findings including organ / body weight ratios:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Microscopic Examination (see also APPENDIX 4)
There were no treatment-related microscopic findings.
One control female (no. 89) had a marked cyst present in the cervix which correlated to the
macroscopic finding in this animal and likely accounted for the infertility. One male rat at 100
mg/kg/day (no. 18) had an extensive bilateral seminiferous tubular atrophy in the testes with a
subsequent extensive epididymal oligospermia, which accounted for its infertility. This was
considered to be a spontaneous abnormality with no likely relationship to the test item.
No other abnormalities were seen in the reproductive organs of the remaining suspected nonfertile
animals which could account for their infertility.
All microscopic findings recorded were considered to be within the normal range of background
pathology encountered in Wistar-Han rats of this age and strain
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of
impaired spermatogenesis.
Macroscopic Examination
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations.
The female at 1000 mg/kg/day euthanized in extremis (no. 117) showed a hard, greenish
nodule on the right medial lobe of the lung, dark red discoloration of the left ovary, enlarged
adrenal glands, reduced size of the thymus, enlargement and greenish discoloration of the
bronchial lymph node and pleura grown together with the lungs.
A watery-clear cyst was found for a single control Repro female (no. 89). This finding was
corroborated with a cyst found in the cervix found upon histopathological examination, which
likely contributed to this animal’s suspected infertility.
Other incidental findings among control and treated animals at the end of the treatment and/or
recovery period included alopecia, red foci on the thymus, reddish discolouration of the thymus
or mesenteric lymph node, pelvic dilation of the kidney, reduced size of the testes,
epididymides or seminal vesicles, yellowish hard nodules, a red-brown focus or tan
discolouration of the clitoral glands, and fluid in the uterus. The incidence of these findings
remained within the background range of findings that are encountered among rats of this age
and strain, and the their incidence did not show a dose-related trend. These necropsy findings
were therefore considered to be of no toxicological relevance.

Effect levels (P0)

Key result

Dose descriptor:

NOAEC

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

other: Microscopic Examination Functional observations

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant clinical signs for any treatment group. Incidental clinical symptoms of pups included small size and scabbing of the neck. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered toxicologically relevant.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One pup of each treatment group was found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
A statistically significant lower mean number of living pups at first litter check observed at 1000 mg/kg/day was due to a low number of pups (5 in total) for one female (no. 111). Since the number of pups of other females of this dose group was within the range considered normal, no toxicological relevance was ascribed to this change.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg, pup body weights were increased on Day 4 of lactation, achieving a level of statistical significance for male pups. Given that this increase was relatively slight in nature, and that the opposite effect would be expected in case of reproductive toxicity, no toxicological relevance was ascribed to these changes.

Details on results (F1)

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Effect levels (F1)

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Any other information on results incl. tables

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Parental results:

No parental toxicity was observed at any dose level.

Reproductive/Developmental results:

No reproduction/developmental toxicity was observed at any dose level.

Applicant's summary and conclusion

Conclusions:

Based on these results, a parental, reproduction and developmental No Observed Adverse
Effect Level (NOAEL) of at least 1000 mg/kg/day was derived.