Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information
Based on the results of the prenatal developmental toxicity study, 1000 mg/kg/day was considered to be the maternal no observed adverse effect level (NOAEL) and the fetal no observed effect level (NOEL).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Experimental results based on internationally recognized OECD 414 study protocol.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Weight range of animals at dosing (230-292g) deviated from target range of 180-250g, and several times temperature and humidity within the animal room deviated from targets. However, deviations had no impact on study integrity.
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Ninety-eight time mated female Crl:CD(SD) Sprague Dawley rats were ordered from Charles River, Margate, Kent, UK and arrived on 10 Jan 2014. Ninety six animals were assigned to the study with the remaining two animals acting as spares.
Animals arrived in 3 sub-batches: one sub-batch was on Day 1 of gestation, the second was on Day 2 of gestation and the third on Day 3 of gestation (day of detection of mating = Day 0 gestation).
At the time of mating the animals were ca 9 weeks old and on arrival weighed 201-255g. At the initiation of dosing, animals weighed 230-292g.

Animals were housed singly in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms.
Bedding material was sterilised white wood shavings which were provided with a certificate of analysis for significant contaminants. An analytical certificate for each batch of bedding used is retained at Charles River, Edinburgh.

Environmental Conditions
The targeted conditions for the animal room environment were as follows:
Temperature: 19 - 23°C.
Humidity: 40-85%.
Ventilation: A minimum of 10 air changes per hour.
Light Cycle: 12 hours dark/12 hours light.
There was automatic control of temperature which was continuously monitored and recorded. Humidity was continuously monitored and recorded. There was automatic control of light cycle. The actual ranges for temperature and humidity were 20-24°C and 33-59%, respectively.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% HPMC (Methocel E4M), 0.1% Tween 80 in Milli-Q water
Details on exposure:
The test and control items were administered to the appropriate rats by once daily oral gavage from Days 6-19 of gestation. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a syringe with attached gavage cannula. The dosing formulations were stirred continuously during dose administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements.
The required amount of test item was weighed and accurately transferred to a pre-labelled container according to instructions from the formulation computerised system (Dispense). The appropriate amount of the vehicle was added to the container and the formulation was magnetically stirred until a homogenous formulation was obtained.
The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and were stirred for at least 30 minutes before dosing and continuously during dosing. Details of the preparation and dispensing of the test item have been retained in the study records.
Dose formulation samples were collected for analysis during weeks 1 and 2 for all groups to confirm concentration and homogeneity. Samples to be analysed were transferred at ambient temperature (<30ºC) to the analytical laboratory at the Test Facility. Analyses were performed by Gas Chromatography with Flame Ionisation Detection using a validated analytical procedure (Charles River Study No. 431130, AP.3113.02).

Samples for analysis: Duplicate top, middle and bottom samples (duplicate middle only for control).
Back-up samples: Triplicate top, middle and bottom samples maintained at the Test Facility. Week 2 back-up samples for Group 2 were analysed. No other back-up analysis was required.
Volume: 1 mL for analysis and back-up samples.
Storage Conditions: 2-8°C, in the dark.
Acceptance Criteria: For concentration, the criteria for acceptability was mean results within or equal to ±10% of theoretical concentration. Each individual sample concentration result within or equal to ±15%. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of ≤10% for each group.
All formulations prepared for use on Weeks 1 and 2 were within the acceptance criteria of ±10% of theoretical concentration, with the exception of Group 2 samples on Week 2, which were found to be 11.6%. Analysis of back-up samples for this group were found to be within specification, therefore it was considered that Group 2 Week 2 samples were suitable for use. Following review of formulation and analytical data, a reason for the initial out of specification results could not be found.
The low relative standard deviation of concentrations on all occasions (<10%) showed that formulation were homogenous.
The absence of test item was confirmed from Control samples.
Duration of treatment / exposure:
The test and control items were administered from Days 6-19 of gestation.
Frequency of treatment:
The test and control items were administered to the appropriate rats by once daily oral gavage during the dosing period.
Duration of test:
The Study Director signed the protocol on 08 Jan 2014, and dosing was initiated on 13 Jan 2014. The in-life phase of the study was completed on 29 Jan 2014. The experimental start date was 10 Jan 2014, and the experimental completion date was 20 Mar 2014.
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
other: control
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
analytical conc.
No. of animals per sex per dose:
Twenty-four animals (twelve of each sex) per dose group, for a total of ninety-six including the control animals. Spare animals were numbered 97 and 98. As no replacements were necessary, these animals were considered not to be part of the study.
Control animals:
yes, concurrent vehicle
Maternal examinations:
All adult animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy examinations were conducted by a trained technician and consisted of an external and internal examination and recording of observations for all animals. A veterinary pathologist was available for consultation during normal working hours. Following necropsy the maternal carcasses were discarded.
Ovaries and uterine content:
The reproductive tract was dissected from the abdominal cavity. The gravid uterus was weighed. The uterus was opened and the contents were examined. The fetuses were removed from the uterus.
The ovaries and uterus were examined for number and distribution of
• Corpora lutea
• Implantation sites
• Placenta (size, colour, shape – only abnormalities were recorded)
• Live and dead foetuses
• Early and late embryonic deaths
Fetal examinations:
External Abnormalities
Foetuses were examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible.
Each implant was classified as being live, or a dead fetus (dead full term fetus that shows no sign of maceration), or a late embryonic death (macerated tissue identifiable as an embryo fetus, with recognisable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placenta), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; which may be of varying size).

Body Weights and Identification
The body weight of each live fetus was recorded and foetuses were individually identified within their litter.

Visceral Examination and Sex
Half of the viable fetuses from each uterus were fixed in methylated ethyl alcohol, the remaining half in Bouin's fluid. Following initial fixation, the fetuses fixed in alcohol were examined by open dissection for abnormalities of the thoracic and abdominal viscera and for sex. These viscera were then discarded. The fetuses fixed in Bouin's fluid were examined for soft tissue abnormalities and sex using a freehand sectioning technique derived from that of Wilson1.

Skeletal Examination
The eviscerated carcasses were then macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification.
Statistics:
Means and standard deviations were calculated for body weight, food consumption and pregnancy data.
Where required to assist interpretations, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Body weight and food consumption data was analysed for homogeneity of variance using the ‘F-max’ test. If the group variances appeared homogenous, a parametric ANOVA was used an pairwise comparisons were made using Fisher’s F protected LSD method via Student’s t test ie pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis nonparametric ANOVA was used and pairwise comparisons were made using chi squared
protection (via z tests, the non-parametric equivalent of Student’s t test).
Fetal weight data was subjected to Kruskal-Wallis non-parametric analysis.
Dixon Q Tests were applied as part of formulation analysis at the discretion of the IS for Formulation Analysis. Details of these tests are retained in the study data and results are presented in the Formulation Analysis Phase Report.
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

RESULTS

Mortality

There were no premature deaths during the study.

Clinical Observations

Observation/Finding

Group/Dose Level (mg/kg/day)

1

2

3

4

(0)

(100)

(300)

(1000)

Clinical Observations

 

 

 

 

Sparse hair

3

1

3

0

Staining on fur

2

2

3

5

Scab(s) on skin

1

0

1

0

Ploughing behaviour

0

0

1

7

Excess salivation

0

0

0

1

Maternal Necropsy Findings

 

 

 

 

Skin – Scab(s)

1

0

0

0

Skin – Staining on fur

2

1

1

1

Skin – Thin haircoat

2

1

1

0

Uterus – Fused placentae

0

1

0

0

Uterus – No visible signs of implantation or countable corpora lutea

0

0

0

1

Number of Females

24

24

24

24

 

At 1000 mg/kg/day, ploughing behaviour (animal burrowing through bedding with its head) was noted in 5/24 animals and excess salivation was noted in one animal.  These observations were transient, being noted immediately post dose and no longer being evident 1 hour after dosing, and were generally noted on only one or two occasions in each individual between Days 15-19 of gestation.  Ploughing behaviour was also noted in one animal at 300 mg/kg/day.

 

All other clinical observations were considered to be incidental background findings commonly observed in this species at Charles River, Edinburgh.

 

Food Consumption

Group mean food consumption was similar in control and treated groups throughout the study.

 

Necropsy Findings

There were no macroscopic necropsy findings in dams or foetuses which were considered to be related to treatment with Alchisor H5.  All findings were considered to be incidental findings and those commonly observed in this species at Charles River, Edinburgh.

 

Pregnancy Performance and Fetal Weights

Pregnancy performance and fetal weights were similar in all groups.  Slight intergroup variations were considered to be incidental and too small to be attributed to treatment with Alchisor H5.

Pregnancy Performance and Fetal Weights (g)

Parameters

Dose Group/Dose Level (mg/kg/day)

1

2

3

4

(0)

(100)

(300)

(1000)

Number of animals mated

24

24

24

24

Number of non-pregnant animals

0

0

0

1

Number pregnant at Day 20 necropsy

24

24

24

23

Pregnancy Frequency as %

100

100

100

96

 

Total corpora lutea graviditatis

355

349

359

338

Total number of implants

340

335

351

319

Pre-implantation loss as %

4

4

2

6

Total live implants (%)

318 (94)

322 (96)

330 (94)

297 (93)

Total dead implants (%)

22 (6)

13 (4)

21 (6)

22 (7)

Total early embryonic deaths (%)

16 (5)

13 (4)

20 (6)

20 (6)

Total late embryonic deaths (%)

6 (2)

0

1 (0.3)

2 (1)

Total fetal deaths (%)

0

0

0

0

Mean corpora lutea graviditatis

14.8 ± 1.5

14.5 ± 1.8

15.0 ± 2.0

14.7 ± 2.0

Mean implants

14.2 ± 2.6

14.0 ± 1.2

14.6 ± 2.2

13.9 ± 2.6

Mean live implants

13.3 ± 3.0

13.4 ± 1.3

13.8 ± 2.6

12.9 ± 2.6

Mean dead implants

0.9 ± 1.6

0.5 ± 0.8

0.9 ± 1.1

1.0 ± 0.9

Mean early embryonic deaths

0.7 ± 1.1

0.5 ± 0.8

0.8 ± 1.1

0.9 ± 0.9

Mean late embryonic deaths

0.3 ± 1.0

0

0.04 ± 0.2

0.1 ± 0.3

Mean fetal deaths

0

0

0

0

Total live male fetuses (%)

154 (48)

156 (48)

169 (51)

149 (50)

Total live female fetuses (%)

164 (52)

166 (52)

161 (49)

148 (50)

Live fetal sex ratio (male:female)

01:01.1

01:01.1

01:01.0

01:01.0

Mean total uterus weight (g)

82 ± 16

82 ± 7

85 ± 14

81 ± 14

Mean litter mean fetal weight (g) †

3.90 ± 0.24

3.89 ± 0.27

3.92 ± 0.17

3.88 ± 0.22

† - Statistically analysed, no statistical significance achieved (p < 0.001).

Non-pregnant animals excluded below the double line.

 

Fetal Abnormalities and Variants

The type and distribution of major and minor fetal abnormalities and skeletal ossification parameters did not indicate any association with treatment.  Slight intergroup differences were considered to be incidental and unrelated to treatment with Alchisor H5.

Group Incidence of Major Fetal Abnormalities

 

Group/Dose Level (mg/kg/day)

 

1

2

3

4

Abnormality

(0)

(100)

(300)

(1000)

 

Incidence of Fetuses (Litters)

Markedly increased subcutaneous spaces head and cervical regions

 

 

 

 

0

0

0

1(1)

Lateral brain ventricles dilated

0

0

0

1(1)

Scapula bent

1(1)

0

0

0

Scapula and radius bent, with humerus shortened and/or bent.

2(1)

0

0

0

Forelimb flexure

0

7(1)

0

0

Right subclavian artery retro-oesophageal

0

0

0

1(1)

Partially duplicated inferior vena cava

0

0

1(1)

0

Sternebra fused. Bilateral rib(s) costal cartilages fused at point of attachment to sternum. Ribs and costal cartilages partially fused.

0

0

0

1(1)

Sternum flattened, with sternebrae connected

1(1)

0

0

0

Abdominal situs inversus

0

1(1)

0

0

Number with major abnormality

4(2)

8(2)

1(1)

4(4)

Total number examined

318(24)

322(24)

330(24)

297(23)

 

Conclusions:
Based on the results of this study, 1000 mg/kg/day was considered to be the maternal no observed adverse effect level (NOAEL) and the fetal no observed effect level (NOEL).
Executive summary:

The objective of this study was to determine the potential toxicity ofAlkenes, C11/C12, Hydroformylation Products, Distillation Residues (CAS/No. 90622-27-8)when the material was administered during the period of organogenesis to pregnant rats.

For brevity, the test item may be referred to as its alternative name “Alchisor H5” where appropriate for the remainder of the report.

The study design was as follows:

 

Experimental Design

Group No.

Number of Animals

Test Item

Dosage Level

(mg/kg/day)

Dosage Concentration

(mg/mL)

Dosage Volume

(mL/kg)

1

24

Control*

0

0

10

2

24

Alchisor H5

100

10

10

3

24

Alchisor H5

300

30

10

4

24

Alchisor H5

1000

100

10

*The control item was 0.5% HPMC (Methocel E4M), 01% Tween 80 in Milli-Q water.

 

All animals were dosed over Days 6-19, inclusive, of gestation, where the day of detection of mating was designated Day 0.

Animals were regularly monitored for clinical signs of toxicity, body weights and food consumption performance and were killed on Day 20 of gestation for examination of pregnancies and embryo-fetal development.

Dosing of Alchisor H5 at dose levels up to 1000 mg/kg/day was not associated with any treatment related effects on body weight or food consumption, or any gross necropsy findings in dams or foetuses.  At 300 and 1000 mg/kg/day, clinical observations of ploughing behaviour and excess salivation were considered not to be adverse due to their transient nature and low incidence.

Pregnancy performance and fetal weights were similar in control and treated groups, and the type and distribution of major fetal abnormalities, minor visceral and skeletal abnormalities and variants and skeletal ossification parameters did not indicate an association with treatment with Alchisor H5.

Based on the results of this study, 1000 mg/kg/day was considered to be the maternal no observed adverse effect level (NOAEL) and the fetal no observed effect level (NOEL).

Effect on developmental toxicity: 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:
well conducted study according to OECD 414 and GLP
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

The objective of this study was to determine the potential toxicity of Alkenes, C11/C12, Hydroformylation Products, Distillation Residues (CAS/No. 90622-27-8)when the material was administered during the period of organogenesis to pregnant rats. For brevity, the test item may be referred to as its alternative name “Alchisor H5” where appropriate.

Dosing of Alchisor H5 at dose levels up to 1000 mg/kg/day was not associated with any treatment related effects on body weight or food consumption, or any gross necropsy findings in dams or foetuses. At 300 and 1000 mg/kg/day, clinical observations of ploughing behaviour and excess salivation were considered not to be adverse due to their transient nature and low incidence.

Pregnancy performance and fetal weights were similar in control and treated groups, and the type and distribution of major fetal abnormalities, minor visceral and skeletal abnormalities and variants and skeletal ossification parameters did not indicate an association with treatment with Alchisor H5.

Based on the results of this study, 1000 mg/kg/day was considered to be the maternal no observed adverse effect level (NOAEL) and the fetal no observed effect level (NOEL).

Justification for classification or non-classification

The available data do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC.