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

Repeated dose toxicity: oral

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Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Repeated dose toxicity,28d study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:Wistar(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

Details on route of administration:

the dose levels for this combined 28-day oral gavage study with
reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000
mg/kg/day.

Vehicle:

polyethylene glycol

Details on oral 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 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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

1

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

Observations and examinations performed and frequency:

Frequency 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.

Other examinations:

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.

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 of examinations

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.
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

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

Description (incidence and severity):

No toxicologically relevant changes were noted in organ weights and organ to body weight
ratios.
Any statistically significant changes in organ weights and organ to body weight ratios 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. Also, no histopathological
correlates were noted to support these changes. These changes consisted of a lower spleen to
body weight ratio in males at 1000 mg/kg/day at the end of the recovery phase, a higher spleen
weight and spleen to body weight ratio in females at 100 mg/kg/day and the end of treatment,
lower heart weight in females at 1000 mg/kg/day, higher heart to body weight ratio in females at
1000 mg/kg/day at the end of treatment, lower adrenal weight and/or adrenal to body weight
ratio in females at 300 and 1000 mg/kg/day at the end of treatment, and a lower ovary and
ovary to body weight ratio in females at 1000 mg/kg/day at the end of treatment.
Other organ weights and organ to body weight ratios among the dose groups were similar to
control levels.

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

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

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.